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Sample records for collecting tubule cells

  1. Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

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    Ishikawa, S.; Okada, K.; Saito, T.

    1988-09-01

    The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ((Ca2+)i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased (Ca2+) significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of (Ca2+)i depended on the intracellular Ca pool, since an AVP-induced rise in (Ca2+)i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased /sup 45/Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells.

  2. Comparative proteomic analysis of kidney distal convoluted tubule and cortical collecting duct cells following long-term hormonal stimulation

    DEFF Research Database (Denmark)

    Wu, Qi; Moller, Hanne; Rosenbaek, Lena Lindtoft

    2017-01-01

    -desamino-8-D-arginine vasopressin (dDAVP, 1nM) or angiotensin II (ANGII, 1nM). Cells were harvested, equally pooled and subjected to offline high-pH fractionation based two dimensional LC-MS/MS analysis (Q-Exactive). Identification and quantification of proteins was performed by MaxQuant. Proteins that had...... FDR threshold in one cell type plus the unique proteins in this cell type. These 1025 mpkDCT specific proteins and 1211 mpkCCD specific proteins under the three conditions were subjected to further bioinformatics analyses including Panther and DAVID gene ontology analyses, E3 ligase...

  3. Thiamine uptake into primary proximal tubule cells and MDCK distal tubule cells; Differential effect of ethanol

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    Pochal, M.A.; Taub, M.; Acara, M. (State Univ. of New York, Buffalo (United States))

    1991-03-11

    Rabbit primary proximal tubule cells (PT) and distal cells from a MDCK cell line (DT) were studied for their ability to accumulate and metabolize {sup 14}C-thiamine and to assess the effect of ethanol on the accumulation. Incubation with 10uM {sup 14}C-thiamine, resulted in a four fold greater accumulation of {sup 14}C in PT compared to DT. Ethanol significantly decreased PT thiamine accumulation to 0.92 {plus minus} 0.09 nmole/mg but had little effect on DT accumulation. Initial thiamine uptake rates were greater in PT than in DT. Ethanol did not produce a significant effect on either initial uptake rate. Ethanol, however, decreased the maximum rate of uptake in PR from 3.20 to 1.75 nmole/mg/min. Although both cell types metabolize {sup 14}C to thiamine phosphates, total amount of metabolite was greater in PT. These data are consistent with cortical slice uptake studies in which thiamine accumulation was associated with its phosphorylation. In these slices both maximal accumulation and metabolism were inhibited by ethanol.

  4. Mechanisms of ion transport in the mesonephric collecting duct system of Bufo bufo as revealed by microelectrode recordings in isolated perfused tubules

    DEFF Research Database (Denmark)

    Møbjerg, Nadja; Larsen, Erik Hviid; Novak, Ivana

    2002-01-01

    amphibian, Ba2+, Bufo bufo, collecting duct, collecting tubule, K+ conductance, K+ secretion, kidney, mesonephros, ouabain, toad......amphibian, Ba2+, Bufo bufo, collecting duct, collecting tubule, K+ conductance, K+ secretion, kidney, mesonephros, ouabain, toad...

  5. K+ transport in the mesonephric collecting duct system of the toad Bufo bufo : microelectrode recordings from isolated and perfused tubules

    DEFF Research Database (Denmark)

    Møbjerg, Nadja; Larsen, Erik Hviid; Novak, Ivana

    2002-01-01

    We studied the mechanisms of K(+) transport in cells from isolated and perfused collecting tubules and ducts from the mesonephric kidney of the toad Bufo bufo. Cells were impaled with microelectrodes across the basal cell membrane. The basolateral membrane potential (V(bl)) depolarized upon chang...

  6. Renal tubule cell repair following acute renal injury.

    Science.gov (United States)

    Humes, H D; Lake, E W; Liu, S

    1995-01-01

    Experimental data suggests the recovery of renal function after ischemic or nephrotoxic acute renal failure is due to a replicative repair process dependent upon predominantly paracrine release of growth factors. These growth factors promote renal proximal tubule cell proliferation and a differentiation phase dependent on the interaction between tubule cells and basement membrane. These insights identify the molecular basis of renal repair and ischemic and nephrotoxic acute renal failure, and may lead to potential therapeutic modalities that accelerate renal repair and lessen the morbidity and mortality associated with these renal disease processes. In this regard, there is a prominent vasoconstrictor response of the renal vasculature during the postischemic period of developing acute renal failure. The intravenous administration of pharmacologic doses of atrial natriuretic factor (ANF) in the postischemic period have proven efficacious by altering renal vascular resistance, so that renal blood flow and glomerular filtration rate improve. ANF also appears to protect renal tubular epithelial integrity and holds significant promise as a therapeutic agent in acute renal failure. Of equal or greater promise are the therapeutic interventions targeting the proliferative reparative zone during the postischemic period. The exogenous administration of epidermal growth factor or insulin-like growth factor-1 in the postischemic period have effectively decreased the degree of renal insufficiency as measured by the peak serum creatinine and has hastened renal recovery as measured by the duration of time required to return the baseline serum creatinine values. A similarly efficacious role for hepatocyte growth factor has also been recently demonstrated.

  7. Proximal Tubule Cell Hypothesis for Cardiorenal Syndrome in Diabetes

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    Akihiko Saito

    2011-01-01

    Full Text Available Incidence of cardiovascular disease (CVD is remarkably high among patients with chronic kidney disease (CKD, even in the early microalbuminuric stages with normal glomerular filtration rates. Proximal tubule cells (PTCs mediate metabolism and urinary excretion of vasculotoxic substances via apical and basolateral receptors and transporters. These cells also retrieve vasculoprotective substances from circulation or synthesize them for release into the circulation. PTCs are also involved in the uptake of sodium and phosphate, which are critical for hemodynamic regulation and maintaining the mineral balance, respectively. Dysregulation of PTC functions in CKD is likely to be associated with the development of CVD and is linked to the progression to end-stage renal disease. In particular, PTC dysfunction occurs early in diabetic nephropathy, a leading cause of CKD. It is therefore important to elucidate the mechanisms of PTC dysfunction to develop therapeutic strategies for treating cardiorenal syndrome in diabetes.

  8. PKB and megalin determine the survival or death of renal proximal tubule cells

    OpenAIRE

    Caruso-Neves, Celso; Pinheiro, Ana Acacia S.; Cai, Hui; Souza-Menezes, Jackson; Guggino, William B.

    2006-01-01

    Renal proximal tubule cells have a remarkable ability to reabsorb large quantities of albumin through megalin-mediated endocytosis. This is an essential process for overall body homeostasis. Overstressing this endocytic system with a prolonged excess of albumin is injurious to proximal tubule cells. How these cells function and protect themselves from injury is unknown. Here, we show that megalin is the sensor that determines whether cells will be protected or injured by albumin. Megalin, thr...

  9. The adult Drosophila malphigian tubules are maintained by multipotent stem cells | Center for Cancer Research

    Science.gov (United States)

    All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified.

  10. Effect of radiologic contrast media on cell volume regulation in rabbit proximal renal tubules.

    Science.gov (United States)

    Galtung, H K; Løken, M; Sakariassen, K S

    2001-05-01

    Most radiographic contrast media are hyperosmotic and able to shrink cells with which they are in contact. The authors studied cell volume control in rabbit proximal renal tubules after incubation with three contrast media: iohexol, ioxaglate, and iodixanol. Proximal renal tubules were isolated from rabbit kidneys. The tubules were exposed to Ringer solutions containing 5% vol/vol iohexol (final osmolality, 330 mOsm), ioxaglate (323 mOsm), iodixanol (305 mOsm), or mannitol (control solutions with identical osmolalities), and tubule volumes were monitored. After 2 hours of incubation, the tubules were stimulated with a hyposmotic Ringer solution (165 mOsm). Three groups of 10 experiments were performed. All solutions induced cell shrinkage (8.3%+/-3.8 [standard error] to 15.4%+/-0.5), which was completely or partly reversible in most experiments (volume increase, 44.8%+/-14.7 to 149.9%+/-107.3) but not those with iohexol and iodixanol. With exposure to the hyposmotic solution, the cells swelled by 11.0%+/-1.8 to 39.7%+/-4.8. In general, the tubules that had been exposed to the most hyperosmotic solution swelled the most. Those exposed to contrast media showed less swelling than the mannitol-exposed controls. In all control experiments, the cells exhibited a gradual shrinkage (43.6%+/-28.5 to 87.0%+/-13). This regulatory response was partly inhibited in tubules exposed to iohexol (39.9%+/-15.8 shrinkage) or iodixanol (8.9%+/-15.8) and completely inhibited in those exposed to ioxaglate. Iohexol and ioxaglate exposure also led to a decrease in water permeability. Exposure to hyperosmotic contrast medium tends to induce prolonged cell shrinkage, decrease the water permeability of the cellular plasma membranes, and compromise the ability to regulate cellular volume. These changes seem to reflect both the hyperosmolality of the solutions and their inherent chemical properties.

  11. Effects of Bacillus thuringiensis kurstaki on Malpighian tubule cells of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) larvae.

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    Ogutchu, Ayşe; Suludere, Zekiye; Uzunhisarcikli, Meltem; Kalender, Yusuf

    2005-01-01

    In this study effects of Bacillus thuringiensis kurstaki (Btk) on Malpighian tubule cells of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae) larvae was investigated by electron microscopy. 3 mg/l Btk was given with food. After Btk administration, the Malpighian tubule cells were investigated and compared with a control group. 3 and 6 hrs after Btk administration swelling in Malpighian tubule cells was observed. Swelling of mitochondria and separation of their cristae was seen after 12 hrs. After 24 hrs dissolution of the basal cytoplasm, swelling and vacuolization of all mitochondria, partial dissolution of the nucleoplasm, and swelling and separation ofmicrovilli was documented. A membrane-body in the nucleus was seen after 48 hrs. The nucleoplasm was completely dissolved after 72 hrs and after 96 hrs large vacuoles appeared in the cytoplasm and shortening of microvilli was observed.

  12. Effect of ADH on rubidium transport in isolated perfused rat cortical collecting tubules

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    Schafer, J.A.; Troutman, S.L.

    1986-06-01

    Unidirectional fluxes of 86Rb+ were measured as an indicator of potassium transport in isolated rat cortical collecting tubules perfused and bathed at 38 degrees C with isotonic solutions in which Rb+ replaced K+. Under control conditions the lumen-to-bath flux (Jl----b) was significantly less than the bath-to-lumen flux (Jb----l), indicating net Rb+ secretion. Net secretion increased approximately 180% after addition of 100 microU/ml of arginine vasopressin (ADH) to the bathing solution, due to a rapid and reversible increase in Jb----l from 4.6 +/- 0.8 to 9.0 +/- 1.9 pmol X min-1 X mm-1 with no significant change in Jl----b. The ADH effect was completely inhibited by 2 mM luminal Ba2+. The average transepithelial voltage (Ve) was not significantly different from zero in the control period but became lumen negative (-5 to -10 mV) after ADH. With 10(-5) M amiloride in the lumen Ve was lumen positive (+2 to +4 mV) and was unaltered by ADH or Ba2+, yet ADH produced a significant but attentuated increase in Jb----l with no change in Jl----b. The results indicate that ADH augments net K+ secretion either by an increase in the Ba2+-sensitive conductance of the apical membrane or by an increase in the electrochemical potential driving force for net Rb+ secretion through this pathway.

  13. Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

    DEFF Research Database (Denmark)

    Møbjerg, Nadja; Larsen, Erik Hviid; Novak, Ivana

    2004-01-01

    It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane...... and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na...

  14. Adipose tissue-derived mesenchymal stem cells repair germinal cells of seminiferous tubules of busulfan-induced azoospermic rats

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    Davood Mehrabani

    2015-01-01

    Full Text Available Context: Adipose tissue-derived mesenchymal stem cells (AT-MSCs are less invasive than bone marrow mesenchymal stem cells to obtain for cell therapy. Aims: The aims of this study were to evaluate the germinal cells characteristics and repairs in seminiferous tubules of busulfan-induced azoospermic rats after AT-MSCs transplantation. Settings and Design: Experimental case-control study. Materials and Methods: In the present experimental study, donors AT-MSCs were isolated from subcutaneous adipose tissue of two Sprague-Dawley rats. The recipients (n = 5 were received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia by busulfan, rats were injected with the AT-MSCs into the efferent duct of right testes. After 60 days, the right testes were injected AT-MSCs were compared to left azoospermic testes. Five untreated male rats served as negative control. Statistical Analysis Used: Stereological indices were analyzed by one-way ANOVA and LSD post-hoc test. The spermatogenesis index was compared using Mann-Whitney U test. Results: After stereological analyses, the seminiferous tubules treated with AT-MSCs had normal morphology. The untreated seminiferous tubules were empty. Spermatogenesis was observed in most cell-treated seminiferous tubules. Conclusions: The testis of busulfan-induced azoospermic rats accepted transplanted AT-MSCs. The transplanted AT-MSCs could induce spermatogenesis in seminiferous tubules of the rat.

  15. Mesenchymal stem cell-conditioned medium accelerates regeneration of human renal proximal tubule epithelial cells after gentamicin toxicity.

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    Moghadasali, Reza; Mutsaers, Henricus A M; Azarnia, Mahnaz; Aghdami, Nasser; Baharvand, Hossein; Torensma, Ruurd; Wilmer, Martijn J G; Masereeuw, Rosalinde

    2013-07-01

    Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity to regenerate renal tubule epithelia and repair renal function without fusing with resident tubular cells. The goal of the present project was to investigate the role of MSCs secreted cytokines on tubule cell viability and regeneration after a toxic insult, using a conditionally immortalized human proximal tubule epithelial cell (ciPTEC) line. Gentamicin was used to induce nephrotoxicity, and cell viability and migration were studied in absence and presence of human MSC-conditioned medium (hMSC-CM) i.e. medium containing soluble factors produced and secreted by MSCs. Exposure of ciPTEC to 0-3000 μg/ml gentamicin for 24 h caused a significant dose-dependent increase in cell death. We further demonstrated that the nephrotoxic effect of 2000 μg/ml gentamicin was recovered partially by exposing cells to hMSC-CM. Moreover, exposure of ciPTEC to gentamicin (1500-3000 μg/ml) for 7 days completely attenuated the migratory capacity of the cells. In addition, following scrape-wounding, cell migration of both untreated and gentamicin-exposed cells was increased in the presence of hMSC-CM, as compared to exposures to normal medium, indicating improved cell recovery. Our data suggest that cytokines secreted by MSCs stimulate renal tubule cell regeneration after nephrotoxicity. Copyright © 2012 Elsevier GmbH. All rights reserved.

  16. Angiotensin II counteracts the effects of cAMP/PKA on NHE3 activity and phosphorylation in proximal tubule cells.

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    Crajoinas, Renato O; Polidoro, Juliano Z; Carneiro de Morais, Carla P A; Castelo-Branco, Regiane C; Girardi, Adriana C C

    2016-11-01

    Binding of angiotensin II (ANG II) to the AT1 receptor (AT1R) in the proximal tubule stimulates Na(+)/H(+) exchanger isoform 3 (NHE3) activity through multiple signaling pathways. However, the effects of ANG II/AT1R-induced inihibitory G protein (Gi) activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. We therefore tested the hypothesis that ANG II reduces cAMP/PKA-mediated phosphorylation of NHE3 on serine 552 and, in doing so, stimulates NHE3 activity. Under basal conditions, ANG II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of the cAMP-elevating agent forskolin (FSK), ANG II blocked FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity, and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of ANG II were blocked by pretreating OKP cells with the AT1R antagonist losartan, highlighting the contribution of the AT1R/Gi pathway in ANG II-mediated NHE3 upregulation under cAMP-elevating conditions. Accordingly, Gi inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of ANG II on NHE3 activity in rat proximal tubules. Collectively, our results suggest that ANG II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway. Moreover, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by ANG II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation.

  17. Far infrared radiation promotes rabbit renal proximal tubule cell proliferation and functional characteristics, and protects against cisplatin-induced nephrotoxicity.

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    Chiang, I-Ni; Pu, Yeong-Shiau; Huang, Chao-Yuan; Young, Tai-Horng

    2017-01-01

    Far infrared radiation, a subdivision of the electromagnetic spectrum, is beneficial for long-term tissue healing, anti-inflammatory effects, growth promotion, sleep modulation, acceleration of microcirculation, and pain relief. We investigated if far infrared radiation is beneficial for renal proximal tubule cell cultivation and renal tissue engineering. We observed the effects of far infrared radiation on renal proximal tubules cells, including its effects on cell proliferation, gene and protein expression, and viability. We also examined the protective effects of far infrared radiation against cisplatin, a nephrotoxic agent, using the human proximal tubule cell line HK-2. We found that daily exposure to far infrared radiation for 30 min significantly increased rabbit renal proximal tubule cell proliferation in vitro, as assessed by MTT assay. Far infrared radiation was not only beneficial to renal proximal tubule cell proliferation, it also increased the expression of ATPase Na+/K+ subunit alpha 1 and glucose transporter 1, as determined by western blotting. Using quantitative polymerase chain reaction, we found that far infrared radiation enhanced CDK5R1, GNAS, NPPB, and TEK expression. In the proximal tubule cell line HK-2, far infrared radiation protected against cisplatin-mediated nephrotoxicity by reducing apoptosis. Renal proximal tubule cell cultivation with far infrared radiation exposure resulted in better cell proliferation, significantly higher ATPase Na+/K+ subunit alpha 1 and glucose transporter 1 expression, and significantly enhanced expression of CDK5R1, GNAS, NPPB, and TEK. These results suggest that far infrared radiation improves cell proliferation and differentiation. In HK-2 cells, far infrared radiation mediated protective effects against cisplatin-induced nephrotoxicity by reducing apoptosis, as indicated by flow cytometry and caspase-3 assay.

  18. A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X in polycystin-1.

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    Brittney-Shea Herbert

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells.

  19. Characterization of the collagen phenotype of rabbit proximal tubule cells in culture.

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    Gibbs, S R; Goins, R A; Belvin, E L; Dimari, S J; Merriam, A P; Bowling-Brown, S; Harris, R C; Haralson, M A

    1999-01-01

    Studies were performed to characterize the collagen phenotype of cultured rabbit proximal tubule (RPT) epithelial cells grown on plastic and on the reconstituted basement membrane preparation, Matrigel. When grown on a plastic substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking network of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of cells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on this substrate. Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SDS-PAGE revealed components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM-Trisacryl chromatography established that approximately 2/3 of the total collagen synthesized by RPT cells grown on plastic was type IV and approximately 1/3 type I. Quantitative analysis of the collagens produced by RPT cells grown on Matrigel again indicated the synthesis of only type IV and type I molecules but in a slightly more equal ratio of both collagen types and in the ratio of secreted to cell-associated molecules. However, the total amount of collagen synthesized by RPT cells grown on Matrigel was reduced to approximately 1% of the level synthesized by the cells grown on plastic. On plastic, approximately 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only approximately 55% of the total type I collagen synthesized. On Matrigel, the majority of the type IV collagen was recovered as heterotrimers containing alpha1(IV) and alpha2(IV) chains. In contrast, RTP cells grown on plastic predominantly produced type IV

  20. Vertebrate kidney tubules elongate using a planar cell polarity-dependent, rosette-based mechanism of convergent extension

    OpenAIRE

    2012-01-01

    Cystic kidney diseases are a global public health burden, affecting over 12 million people1. Although much is known about the genetics of kidney development and disease, the cellular mechanisms driving normal kidney tubule elongation remain unclear 2,3. Here, we used in vivo imaging to demonstrate for the first time that mediolaterally-oriented cell intercalation is fundamental to vertebrate kidney morphogenesis. Surprisingly, kidney tubule elongation is driven in large part by a myosin-depen...

  1. A bioartificial renal tubule device embedding human renal stem/progenitor cells.

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    Anna Giovanna Sciancalepore

    Full Text Available We present a bio-inspired renal microdevice that resembles the in vivo structure of a kidney proximal tubule. For the first time, a population of tubular adult renal stem/progenitor cells (ARPCs was embedded into a microsystem to create a bioengineered renal tubule. These cells have both multipotent differentiation abilities and an extraordinary capacity for injured renal cell regeneration. Therefore, ARPCs may be considered a promising tool for promoting regenerative processes in the kidney to treat acute and chronic renal injury. Here ARPCs were grown to confluence and exposed to a laminar fluid shear stress into the chip, in order to induce a functional cell polarization. Exposing ARPCs to fluid shear stress in the chip led the aquaporin-2 transporter to localize at their apical region and the Na(+K(+ATPase pump at their basolateral portion, in contrast to statically cultured ARPCs. A recovery of urea and creatinine of (20±5% and (13±5%, respectively, was obtained by the device. The microengineered biochip here-proposed might be an innovative "lab-on-a-chip" platform to investigate in vitro ARPCs behaviour or to test drugs for therapeutic and toxicological responses.

  2. Myelin-like structures seen intracellularly in renal tubule cells subjected to ischemia.

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    Yamada,Teruo

    1980-02-01

    Full Text Available Renal cortex was studied during experimentally induced ischemia. A transient increase in anerobic glycolysis occurred with concomitant swelling of both the Golgi apparatus and mitochondria. These intracytoplasmic organelles underwent marked changes in their intracellular positions. Infolding of cytoplasmic membrane at the basal side of proximal tubule cells increased in complexity and proceeded to enclose various intracytoplasmic microorganelles such as mitochondria and the Golgi apparatus. Piling up in layers was particularly marked around mitochondria. This piling up appeared as myelin-like structures on the free surface of, and within, proximal tubule cells, and followed disruption of the brush border at the free surface. Histological examination of thin sections showed that the fused portions of this brush border were actually brush border cytoplasmic membrane piled up in layers giving the appearance of myelin-like structures. After two hours of ischemia, parts of the membrane of these myelin-like structures were disrupted. Large vacuoles developed and these were thought to be related to the large vacuoles seen during cell degeneration.

  3. Dopamine and angiotensin type 2 receptors cooperatively inhibit sodium transport in human renal proximal tubule cells.

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    Gildea, John J; Wang, Xiaoli; Shah, Neema; Tran, Hanh; Spinosa, Michael; Van Sciver, Robert; Sasaki, Midori; Yatabe, Junichi; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2012-08-01

    Little is known regarding how the kidney shifts from a sodium and water reclaiming state (antinatriuresis) to a state where sodium and water are eliminated (natriuresis). In human renal proximal tubule cells, sodium reabsorption is decreased by the dopamine D(1)-like receptors (D(1)R/D(5)R) and the angiotensin type 2 receptor (AT(2)R), whereas the angiotensin type 1 receptor increases sodium reabsorption. Aberrant control of these opposing systems is thought to lead to sodium retention and, subsequently, hypertension. We show that D(1)R/D(5)R stimulation increased plasma membrane AT(2)R 4-fold via a D(1)R-mediated, cAMP-coupled, and protein phosphatase 2A-dependent specific signaling pathway. D(1)R/D(5)R stimulation also reduced the ability of angiotensin II to stimulate phospho-extracellular signal-regulated kinase, an effect that was partially reversed by an AT(2)R antagonist. Fenoldopam did not increase AT(2)R recruitment in renal proximal tubule cells with D(1)Rs uncoupled from adenylyl cyclase, suggesting a role of cAMP in mediating these events. D(1)Rs and AT(2)Rs heterodimerized and cooperatively increased cAMP and cGMP production, protein phosphatase 2A activation, sodium-potassium-ATPase internalization, and sodium transport inhibition. These studies shed new light on the regulation of renal sodium transport by the dopaminergic and angiotensin systems and potential new therapeutic targets for selectively treating hypertension.

  4. Collective cell migration drives morphogenesis of the kidney nephron.

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    Aleksandr Vasilyev

    2009-01-01

    Full Text Available Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase-positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow-dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.

  5. Entosis Acts as a Novel Way within Sertoli Cells to Eliminate Spermatozoa in Seminiferous Tubule

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    Nisar Ahmed

    2017-05-01

    Full Text Available The present study was designed to investigate the hypothesis that in vivo entosis is a novel pathway for eliminating spermatozoa in the seminiferous tubules (ST during hibernation of the Chinese soft-shelled turtle. Western blot analysis revealed that the expression of LAMP1 in the testis was significantly higher during hibernation than that during non-hibernation. Immunohistochemistry reaction showed that LAMP1-positive substance was distributed within the Sertoli cells of the testis. Further examination by transmission electron microscopy (TEM, many degraded spermatozoa being enwrapped within large entotic vacuoles in Sertoli cells. The nucleus and the flagellum of the spermatozoa were shown to be decomposed and digested inside entotic vacuoles within Sertoli cells. More than two spermatozoa heads were always observed in each internalized vacuoles. Deserving note is that, a number of different autophagosomes, including initial autophagic vesicles and degradative autophagic vesicles were found inside the entotic vacuoles of the Sertoli cells during hibernation. At the end of hibernation, entotic vacuoles and their autophagosomes disappeared, and numerous large lipid droplets (LDs appeared within the Sertoli cells. Adherens junctions were apparent between Sertoli cells and developing germ cells, which is the ultrastructural basis of entosis. Taken together, the results presented here show that in the turtle: (1 entosis with internal autophagosomes can take place within normal body cells during hibernation; (2 spermatozoa, as a highly differentiated cell can be internalized and degraded within Sertoli cell by entosis in vivo, which is in favor of the next reproductive cycle in the turtle.

  6. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Sam Coffey

    Full Text Available Diabetes mellitus (DM has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160 and cytoplasmic tail of megalin. Mice with type 1 DM (T1D displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN at an earlier stage.

  7. Akt Links Insulin Signaling to Albumin Endocytosis in Proximal Tubule Epithelial Cells.

    Science.gov (United States)

    Coffey, Sam; Costacou, Tina; Orchard, Trevor; Erkan, Elif

    2015-01-01

    Diabetes mellitus (DM) has become an epidemic, causing a significant decline in quality of life of individuals due to its multisystem involvement. Kidney is an important target organ in DM accounting for the majority of patients requiring renal replacement therapy at dialysis units. Microalbuminuria (MA) has been a valuable tool to predict end-organ damage in DM but its low sensitivity has driven research efforts to seek other alternatives. Albumin is taken up by albumin receptors, megalin and cubilin in the proximal tubule epithelial cells. We demonstrated that insulin at physiological concentrations induce albumin endocytosis through activation of protein kinase B (Akt) in proximal tubule epithelial cells. Inhibition of Akt by a phosphorylation deficient construct abrogated insulin induced albumin endocytosis suggesting a role for Akt in insulin-induced albumin endocytosis. Furthermore we demonstrated a novel interaction between Akt substrate 160kDa (AS160) and cytoplasmic tail of megalin. Mice with type 1 DM (T1D) displayed decreased Akt, megalin, cubilin and AS160 expression in their kidneys in association with urinary cubilin shedding preceding significant MA. Patients with T1D who have developed MA in the EDC (The Pittsburgh Epidemiology of Diabetes Complications) study demonstrated urinary cubilin shedding prior to development of MA. We hypothesize that perturbed insulin-Akt cascade in DM leads to alterations in trafficking of megalin and cubilin, which results in urinary cubilin shedding as a prelude to MA in early diabetic nephropathy. We propose that utilization of urinary cubilin shedding, as a urinary biomarker, will allow us to detect and intervene in diabetic nephropathy (DN) at an earlier stage.

  8. In utero exposure to diethylstilboestrol or 4-n-nonylphenol in rats: Number of Sertoli cells, diameter and length of seminiferous, tubules estimated by stereological methods

    DEFF Research Database (Denmark)

    Dalgaard, Majken; Pilegaard, Kirsten; Ladefoged, Ole

    2002-01-01

    of seminiferous tubules, and the number of Sertoli cells were investigated with stereological methods. Such unbiased methods have not previously been applied on testis diameter and length or on Sertoli cell number of 11-day-old rats. In the control group, the mean length of the seminiferous tubule was 3,0 m+/-0...

  9. Comparison of hyaluronidase expression, invasiveness and tubule formation promotion in ER (-) and ER (+) breast cancer cell lines in vitro

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-yi; TAN Jin-xiang; Marc Vasse; Bertrand Delpech; REN Guo-sheng

    2009-01-01

    Background Hyaluronidase (Hyase) is an enzyme which hydrolyses hyaluronan (HA), a large nonsulfated glycosaminoglycan. Several genes have been identified to code for hyaluronidases in humans. Its role has only recently been underlined in the invasion of prostate cancer, colonic cancer, and breast cancer. Moreover, the findings were in agreement with some experimental results which showed that HA-derived oligosaccharides had angiogenesis-promoting activity. All these findings prompted us to investigate factors that had been characterized as putative invasive factors in different human breast cancer-derived cell lines.Methods We selected two series of human breast cancer-derived cell lines whose expression of estrogen receptors (ER) was previously published. Hyaluronidase secretion in culture medium and expression of matrix metallo-proteinase (MMP)-9, cathepsin-D (cath-D) and vascular endothelial growth factor (VEGF) by cells were determined. We also investigated cell invasiveness in the Matrigel invasion assay, and studied the capability of cancer cells to promote in vitro formation of tubules by endothelial cells.Results ER(-) cells secreted significantly more hyaluronidase (P <0.001) and expressed significantly more VEGF (P <0.01), MMP-9 (P <0.05) and cath-D (P <0.0001) than ER(+) cells. Invasion through Matdgel by ER(-) Hyase(+) cells was significantly higher than that by ER(+) Hyase(-) cells (P<0.05). In both cases, invasion was decreased by heparin (P <0.05). When ECV-304 endothelial cells were co-cultivated in millicell chambers with cancer cells, ECV-304 cells were induced to form tubules. Tubule formation was demonstrated to be more prominent with ER(-) Hyase(+) cells than with ER(+) Hyase(-) cells (P <0.05).Conclusion Invasive features of ER(-) breast cancer cells can be characterized in vitro by an invasive Matrigel assay,as the induction of tubule formation by ECV-304 endothelial cells, higher secretion of hyaluronidase, and higher expression of

  10. 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...

  11. Fluid shear stress increases transepithelial transport of Ca2+ in ciliated distal convoluted and connecting tubule cells.

    NARCIS (Netherlands)

    Mohammed, S.G.; Arjona, F.J.; Latta, F.; Bindels, R.J.M.; Roepman, R.; Hoenderop, J.G.J.

    2017-01-01

    In kidney, transcellular transport of Ca2+ is mediated by transient receptor potential vanilloid 5 and Na+-Ca2+ exchanger 1 proteins in distal convoluted and connecting tubules (DCT and CNT, respectively). It is not yet understood how DCT/CNT cells can adapt to differences in tubular flow rate and,

  12. Short term exposure to elevated levels of leptin reduces proximal tubule cell metabolic activity.

    Science.gov (United States)

    Briffa, Jessica F; Grinfeld, Esther; McAinch, Andrew J; Poronnik, Philip; Hryciw, Deanne H

    2014-01-25

    Leptin plays a pathophysiological role in the kidney, however, its acute effects on the proximal tubule cells (PTCs) are unknown. In opossum kidney (OK) cells in vitro, Western blot analysis identified that exposure to leptin increases the phosphorylation of the mitogen-activated protein kinase (MAPK) p44/42 and the mammalian target of rapamycin (mTOR). Importantly leptin (0.05, 0.10, 0.25 and 0.50 μg/ml) significantly reduced the metabolic activity of PTCs, and significantly decreased protein content per cell. Investigation of the role of p44/42 and mTOR on metabolic activity and protein content per cell, demonstrated that in the presence of MAPK inhibitor U0126 and mTOR inhibitor Ku-63794, that the mTOR pathway is responsible for the reduction in PTC metabolic activity in response to leptin. However, p44/42 and mTOR play no role the reduced protein content per cell in OKs exposed to leptin. Therefore, leptin modulates metabolic activity in PTCs via an mTOR regulated pathway.

  13. Acute leptin exposure reduces megalin expression and upregulates TGFβ1 in cultured renal proximal tubule cells.

    Science.gov (United States)

    Briffa, Jessica F; Grinfeld, Esther; Mathai, Michael L; Poronnik, Phillip; McAinch, Andrew J; Hryciw, Deanne H

    2015-02-05

    Increased leptin concentrations observed in obesity can lead to proteinuria, suggesting that leptin may play a role in obesity-related kidney disease. Obesity reduces activation of AMP-activated protein kinase (AMPK) and increases transforming growth factor-β1 (TGF-β1) expression in the kidney, leading to albuminuria. Thus we investigated if elevated leptin altered AMPK and TGF-β1 signaling in proximal tubule cells (PTCs). In opossum kidney (OK) PTCs Western blot analysis demonstrated that leptin upregulates TGF-β1 secretion (0.50 µg/ml) and phosphorylated AMPKα (at 0.25, and 0.50 µg/ml), and downregulates megalin expression at all concentrations (0.05-0.50 µg/ml). Using the AMPK inhibitor, Compound C, leptin exposure regulated TGF-β1 expression and secretion in PTCs via an AMPK mediated pathway. In addition, elevated leptin exposure (0.50 µg/ml) reduced albumin handling in OK cells independently of megalin expression. This study demonstrates that leptin upregulates TGF-β1, reduces megalin, and reduces albumin handling in PTCs by an AMPK mediated pathway.

  14. MDR1 transporter protects against paraquat-induced toxicity in human and mouse proximal tubule cells.

    Science.gov (United States)

    Wen, Xia; Gibson, Christopher J; Yang, Ill; Buckley, Brian; Goedken, Michael J; Richardson, Jason R; Aleksunes, Lauren M

    2014-10-01

    Paraquat is a herbicide that is highly toxic to the lungs and kidneys following acute exposures. Prior studies have demonstrated that the organic cation transporter 2 and multidrug and toxin extrusion protein 1 contribute to the urinary secretion of paraquat in the kidneys. The purpose of this study was to determine whether the multidrug resistance protein 1 (MDR1/Mdr1, ABCB1, or P-glycoprotein) also participates in the removal of paraquat from the kidneys and protects against renal injury. Paraquat transport and toxicity were quantified in human renal proximal tubule epithelial cells (RPTEC) that endogenously express MDR1, HEK293 cells overexpressing MDR1, and Mdr1a/1b knockout mice. In RPTEC cells, reduction of MDR1 activity using the antagonist PSC833 or siRNA transfection increased the cellular accumulation of paraquat by 50%. Reduced efflux of paraquat corresponded with enhanced cytotoxicity in PSC833-treated cells. Likewise, stable overexpression of the human MDR1 gene in HEK293 cells reduced intracellular levels of paraquat by 50%. In vivo studies assessed the renal accumulation and subsequent nephrotoxicity of paraquat (10 or 30 mg/kg ip) in wild-type and Mdr1a/1b knockout mice. At 4 h after paraquat treatment, renal concentrations of paraquat in the kidneys of Mdr1a/1b knockout mice were 750% higher than wild-type mice. By 72 h, paraquat-treated Mdr1a/1b knockout mice had more extensive tubular degeneration and significantly greater mRNA expression of kidney injury-responsive genes, including kidney injury molecule-1, lipocalin-2, and NAD(P)H quinone oxidoreductase 1, compared with wild-type mice. In conclusion, MDR1/Mdr1 participates in the elimination of paraquat from the kidneys and protects against subsequent toxicity.

  15. In vitro safety assessment of food ingredients in canine renal proximal tubule cells.

    Science.gov (United States)

    Koči, J; Jeffery, B; Riviere, J E; Monteiro-Riviere, N A

    2015-03-01

    In vitro models are useful tools to initially assess the toxicological safety hazards of food ingredients. Toxicities of cinnamaldehyde (CINA), cinnamon bark oil, lemongrass oil (LGO), thymol, thyme oil (TO), clove leaf oil, eugenol, ginger root extract (GRE), citric acid, guanosine monophosphate, inosine monophosphate and sorbose (SORB) were assessed in canine renal proximal tubule cells (CPTC) using viability assay and renal injury markers. At LC50, CINA was the most toxic (0.012mg/ml), while SORB the least toxic (>100mg/ml). Toxicities (LC50) of positive controls were as follows: 4-aminophenol (0.15mg/ml in CPTC and 0.083mg/ml in human PTC), neomycin (28.6mg/ml in CPTC and 27.1mg/ml in human PTC). XYL displayed lowest cytotoxic potency (LC50=82.7mg/ml in CPTC). In vivo renal injury markers in CPTC were not significantly different from controls. The LGO toxicity mechanism was analyzed using qPCR and electron microscopy. Out of 370 genes, 57 genes (15.4%) were significantly up (34, 9.1%) or down (23, 6.2%) regulated, with the most upregulated gene gsta3 (∼200-fold) and the most affected pathway being oxidative stress. LGO induced damage of mitochondria, phospholipid accumulation and lack of a brush border. Viability assays along with mechanistic studies in the CPTC model may serve as a valuable in vitro toxicity screening tool.

  16. The Endocytic Receptor Megalin and its Associated Proteins in Proximal Tubule Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Shankhajit De

    2014-07-01

    Full Text Available Receptor-mediated endocytosis in renal proximal tubule epithelial cells (PTECs is important for the reabsorption and metabolization of proteins and other substances, including carrier-bound vitamins and trace elements, in glomerular filtrates. Impairment of this endocytic process results in the loss of such substances and development of proteinuria, which is an important clinical indicator of kidney diseases and is also a risk marker for cardiovascular disease. Megalin, a member of the low-density lipoprotein receptor gene family, is a multiligand receptor expressed in the apical membrane of PTECs and plays a central role in the endocytic process. Megalin interacts with various intracellular adaptor proteins for intracellular trafficking and cooperatively functions with other membrane molecules, including the cubilin-amnionless complex. Evidence suggests that megalin and the cubilin-amnionless complex are involved in the uptake of toxic substances into PTECs, which leads to the development of kidney disease. Studies of megalin and its associated molecules will be useful for future development of novel strategies for the diagnosis and treatment of kidney diseases.

  17. Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Peiying Yu

    2014-01-01

    Full Text Available NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5 gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT and hypertensive subjects (HT. We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2±0.7-fold, 5.2±0.7-fold, and 2.8±0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5±0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2±0.2-fold greater in HT than NT (P=<0.001, n=6–14/group. Ionomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n=4, ANOVA, effects that were abrogated by pre-treatment of the RPT cells with diphenylene-iodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (−45.1±3.2% vs. mock-siRNA, n=6–8 in HT. D1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT (−32.5±1.8% than HT (−14.8±1.8. In contrast to the marked increase in expression and activity of NOX5 in HT, NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system.

  18. Immunohistochemical Characterization of Spontaneous Sertoli Cell Clusters in the Seminiferous Tubules of C57BL/6J Mice.

    Science.gov (United States)

    Anagawa-Nakamura, Akiko; Kakimoto, Kochi; Miyajima, Katsuhiro; Yasui, Yuzo; Kemmochi, Yusuke; Toyoda, Kaoru; Taniai, Eriko; Takahashi, Akemi; Shoda, Toshiyuki

    2015-07-01

    Cell clusters were observed in the seminiferous tubules of C57BL/6J mice as a spontaneous lesion in a 2-week toxicity study, and they were demonstrated to be basically composed of Sertoli cells by immunohistochemistry for claudin-11 and GATA-4 (GATA-binding protein 4), which are both Sertoli cell markers. The clusters were composed of about 5 to 50 cells, which had eosinophilic and occasionally vacuolated cytoplasm with an unclear cell boundary. The cell clusters involved some sperm. No mitotic figures were observed and no immunoreactivity for proliferating cell nuclear antigen (PCNA) was detected in the clusters. In most cases, the cell clusters were observed in seminiferous tubules that also showed degenerative changes. In rare instances, cell aggregates immunohistochemically positive for claudin-11 were observed in the lumen of the epididymis, suggesting that some of the Sertoli cell clusters were sloughed off from the seminiferous epithelium into the epididymal ducts. To our knowledge, this is the first report of Sertoli cell clusters in any animal species except for transgenic or surgically altered animals. © 2015 by The Author(s).

  19. Effect of radiologic contrast material on cell volume regulation in proximal renal tubules from trout (Salmo trutta).

    Science.gov (United States)

    Galtung, H K; Løken, M; Sakariassen, K S

    2000-11-01

    Most radiographic contrast media (CM) are hyperosmotic and pose an osmotic threat to cells they are in contact with. To study these effects at the cellular level, cell volume regulatory mechanisms were observed in proximal renal tubules following exposure to the CM iohexol, ioxaglate, and iodixanol. Isolated renal tubules from trout (Salmo trutta) were exposed to 5% vol/vol iohexol (326 mOsm), ioxaglate (314 mOsm), or iodixanol (300 mOsm) or mannitol (to achieve the same osmolalities), and cell volume changes were observed videometrically. Iohexol and ioxaglate solutions induced a rapid shrinkage (12%-13%) not followed by cell volume regulation. Without CM (same osmolality), the cells shrank 11% but then showed a 77%-88% volume recovery. This reswelling was inhibited by 55% with the Na+, K+, Cl- symporter inhibitor bumetanide (50 micromol/L). Iodixanol did not significantly affect cell volume. Tubules preincubated with CM or mannitol were then stimulated with a hypoosmotic Ringer solution (160 mOsm) resulting in a 26%-36% cellular volume increase. Compared with results of experiments without mannitol and CM, preexposure to iohexol or ioxaglate almost completely inhibited the expected regulatory shrinkage phase, while previous exposure to hyperosmotic solutions with mannitol reduced the shrinkage response by 40%-53%. In this system, the hyperosmotic iohexol and ioxaglate cause cell shrinkage followed by an impaired cell volume regulatory response. Exposure to these two CM also inhibits cell volume regulation on hypoosmotic stimulation. The isosmotic iodixanol has no such effects. These changes appear to some extent to be a result of the CM's degree of hyperosmolality, but this property alone does not explain these findings.

  20. Silencing megalin and cubilin genes inhibits myeloma light chain endocytosis and ameliorates toxicity in human renal proximal tubule epithelial cells.

    Science.gov (United States)

    Li, Min; Balamuthusamy, Saravanan; Simon, Eric E; Batuman, Vecihi

    2008-07-01

    Using target-specific short interfering (si) RNAs, we silenced the tandem endocytic receptors megalin and cubilin genes in cultured human renal proximal tubule epithelial cells. Transfection by siRNA resulted in up to 90% suppression of both megalin and cubilin protein and mRNA expression. In HK-2 cells exposed to kappa-light chain for up to 24 h, light chain endocytosis was reduced in either megalin- or cubilin-silenced cells markedly but incompletely. Simultaneous silencing of both the cubilin and megalin genes, however, resulted in near-complete inhibition of light chain endocytosis, as determined by measuring kappa-light chain protein concentration in cell cytoplasm and by flow cytometry using FITC-labeled kappa-light chain. In these cells, light chain-induced cytokine responses (interleukin-6 and monocyte chemoattractant protein-1) and epithelial-to-mesenchymal transition as well as the associated cellular and morphological alterations were also markedly suppressed. The results demonstrate that light chain endocytosis is predominantly mediated by the megalin-cubilin tandem endocytic receptor and identify endocytosis as a key step in light chain cytotoxicity. Blocking light chain endocytosis prevents its nephrotoxic effects on human kidney proximal tubule cells.

  1. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

    Directory of Open Access Journals (Sweden)

    Daniel C. Chung

    2011-11-01

    Full Text Available Background/Aims: Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods: Adeno-associated virus (AAV offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber’s congenital amaurosis, a disease of childhood blindness. Results: Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions: We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.

  2. Features of impaired seminiferous tubule differentiation are associated with germ cell neoplasia in adult men surgically treated in childhood because of cryptorchidism.

    Directory of Open Access Journals (Sweden)

    Marek Sosnowski

    2008-04-01

    Full Text Available Seminiferous tubule differentiation was related to the occurrence of germ cell neoplasia in 38 men, aged 17-47, treated surgically in childhood for cryptorchidism. Tissues from 46 testes obtained from biopsies taken as a neoplastic preventive procedure or whole testes removed because of GCT were evaluated quantitatively. Paraffin sections were treated with antibodies against placental like alkaline phosphatase (PLAP, a marker of germ cell neoplasia, and cytokeratin 18 (CK-18, a marker of immature Sertoli cells. Quality of spermatogenesis and number Leydig cells were assessed with a score count. Seminiferous tubules diameter, thickness of basal membrane and size of intertubular spaces were measured with image analysis software. In 17.4% of testes spermatogenesis was normal (9.9 points (N and neoplasia was not found there. In the other 38 specimens (83% spermatogenesis was abnormal (A. When spermatogenesis was arrested or when germ cells were absent (3.7+/-1.8 points, neoplastic lesions were found in 13.1% of the specimens. In A group 5.1+/-7.1% of tubules contained immature Sertoli cells, while in N they were not found. Tubular diameter was significantly lower in A (161.5+/-31.8 microm than in N (184.6+/-24.3 microm and the percentage of seminiferous tubules with the thickening of tubular basal membrane was also greater in A. Intertubular spaces were significantly larger in A (49.9+/-18.6% in comparison to N group (32.6+/-12.5%. Mean number of Leydig cells was similar in both groups. To conclude, in most of the formerly cryptorchid testes, despite surgical treatment, impaired seminiferous tubules differentiation is predominant. Germ cell neoplasia is present in testes with retarded seminiferous tubules differentiation. Retardation of seminiferous tubule differentiation consists of inhibited spermatogenesis, presence of tubules with immature Sertoli cells, decreased tubular diameter, increased thickness of basal membrane and enlarged intertubular

  3. 'Special K' and a Loss of Cell-To-Cell Adhesion in Proximal Tubule-Derived Epithelial Cells: Modulation of the Adherens Junction Complex by Ketamine

    Science.gov (United States)

    Hills, Claire E.; Jin, Tianrong; Siamantouras, Eleftherios; Liu, Issac K-K; Jefferson, Kieran P.; Squires, Paul E.

    2013-01-01

    Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention. PMID:24009666

  4. Activation of D4 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells.

    Science.gov (United States)

    Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D; Jose, Pedro A; Zeng, Chunyu

    2015-01-01

    The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal angiotensin type 1 (AT1) receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto (WKY) rats, but the D4 receptor regulation of AT1 receptor is aberrant in renal proximal tubule cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time- and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na(+)-K(+) ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na(+)-K(+) ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na(+)-K(+) ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na(+)-K(+) ATPase activity in SHR cells. This was confirmed in vivo; pretreatment with PD128077 for 1 week augmented the antihypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension.

  5. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis.

    Science.gov (United States)

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J; Lipschutz, Joshua H

    2014-12-15

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.

  6. A New Look at Electrolyte Transport in the Distal Tubule

    OpenAIRE

    Eladari, Dominique; Chambrey, Régine; Peti-Peterdi, Janos

    2011-01-01

    The distal nephron plays a critical role in the renal control of homeostasis. Until very recently most studies focused on the control of Na+, K+, and water balance by principal cells of the collecting duct and the regulation of solute and water by hormones from the renin-angiotensin-aldosterone system and by antidiuretic hormone. However, recent studies have revealed the unexpected importance of renal intercalated cells, a subtype of cells present in the connecting tubule and collecting ducts...

  7. Novel roles for ceramides, calpains and caspases in kidney proximal tubule cell apoptosis: lessons from in vitro cadmium toxicity studies.

    Science.gov (United States)

    Lee, Wing-Kee; Thévenod, Frank

    2008-12-01

    Apoptosis is a tightly regulated physiological process, which can be initiated by toxic stimuli, such as cadmium (Cd2+). Cd2+ (10-50 microM) induces a rapid increase in reactive oxygen species (ROS) (> or = 30 min) in a cell line derived from the S1 segment of rat kidney proximal tubule, without any apparent mitochondrial dysfunction. The sphingolipid ceramide is an important second messenger in apoptosis. Short exposure to Cd2+ (3h) causes an increase in ceramides, which occurs downstream of ROS formation, and may interact with cellular components, such as endoplasmic reticulum and mitochondria. Following apoptosis initiation, execution must take place. The classical executioners of apoptosis are caspases, a family of cysteine proteases. However, increasing studies report caspase-independent apoptosis, which questions the essentiality of caspases for apoptosis implementation. With low micromolar Cd2+ concentrations (calpains, has emerged. Calpain activation by Cd2+ (3-6h) seems to be regulated by ceramide levels, in order to induce apoptosis. Calpain and caspase substrates overlap but yield different fragments, which may explain their diverse downstream targets. Furthermore, calpains and caspases may interact with one another to enhance, as seen by Cd2+, or diminish apoptosis. In this review, we discuss novel roles for ceramides, calpains and caspases as part of Cd2+-induced apoptotic signalling pathways in the kidney proximal tubule and their in vivo relevance to Cd2+-induced nephrotoxicity.

  8. CD8(+) T cells stimulate Na-Cl co-transporter NCC in distal convoluted tubules leading to salt-sensitive hypertension.

    Science.gov (United States)

    Liu, Yunmeng; Rafferty, Tonya M; Rhee, Sung W; Webber, Jessica S; Song, Li; Ko, Benjamin; Hoover, Robert S; He, Beixiang; Mu, Shengyu

    2017-01-09

    Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention and salt-sensitive hypertension is unknown. Here we demonstrate that T cells infiltrate into the kidney of salt-sensitive hypertensive animals. In particular, CD8(+) T cells directly contact the distal convoluted tubule (DCT) in the kidneys of DOCA-salt mice and CD8(+) T cell-injected mice, leading to up-regulation of the Na-Cl co-transporter NCC, p-NCC and the development of salt-sensitive hypertension. Co-culture with CD8(+) T cells upregulates NCC in mouse DCT cells via ROS-induced activation of Src kinase, up-regulation of the K(+) channel Kir4.1, and stimulation of the Cl(-) channel ClC-K. The last event increases chloride efflux, leading to compensatory chloride influx via NCC activation at the cost of increasing sodium retention. Collectively, these findings provide a mechanism for adaptive immunity involvement in the kidney defect in sodium handling and the pathogenesis of salt-sensitive hypertension.

  9. CD8+ T cells stimulate Na-Cl co-transporter NCC in distal convoluted tubules leading to salt-sensitive hypertension

    Science.gov (United States)

    Liu, Yunmeng; Rafferty, Tonya M.; Rhee, Sung W.; Webber, Jessica S.; Song, Li; Ko, Benjamin; Hoover, Robert S.; He, Beixiang; Mu, Shengyu

    2017-01-01

    Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention and salt-sensitive hypertension is unknown. Here we demonstrate that T cells infiltrate into the kidney of salt-sensitive hypertensive animals. In particular, CD8+ T cells directly contact the distal convoluted tubule (DCT) in the kidneys of DOCA-salt mice and CD8+ T cell-injected mice, leading to up-regulation of the Na-Cl co-transporter NCC, p-NCC and the development of salt-sensitive hypertension. Co-culture with CD8+ T cells upregulates NCC in mouse DCT cells via ROS-induced activation of Src kinase, up-regulation of the K+ channel Kir4.1, and stimulation of the Cl− channel ClC-K. The last event increases chloride efflux, leading to compensatory chloride influx via NCC activation at the cost of increasing sodium retention. Collectively, these findings provide a mechanism for adaptive immunity involvement in the kidney defect in sodium handling and the pathogenesis of salt-sensitive hypertension. PMID:28067240

  10. Maturation, proliferation and apoptosis of seminal tubule cells at puberty after administration of estradiol, follicle stimulating hormone or both

    Institute of Scientific and Technical Information of China (English)

    Renata Walczak-Jedrzejowska; Jolanta Slowikowska-Hilczer; Katarzyna Marchlewska; Krzysztof Kula

    2008-01-01

    Aim: To assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone (FSH) or both agents given together. Methods: From postnatal day (PND) 5 to 15 male rats were daily injected with 12.5 μg of 17β-estradiol benzoate (EB) or 7.5 IU of human purified FSH (hFSH) or EB + hFSH or solvents (control). On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the semini- ferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUNEL method. Results: Although EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB + hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB + hFSH inhibited Sertoli cell proliferation. All treatments signifi- cantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB + hFSH. Conclusion: At puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis. (Asian J Androl 2008 Jul; 10: 585-592)

  11. 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

    homeostasis occurs predominantly via changes in the activity of the high-affinity taurine transport system by alterations in the uptake capacity and with an unaffected half-saturation constant. An adaptive response was not observed for the structurally related beta-alanine. 3. Only colchicine, which......), mimicking the effects of diacylglycerol, induced inhibition of both beta-alanine and taurine uptake. By contrast, the Ca2(+)-ionophore A23187, mimicking the effects of IP3, only stimulated the uptake of taurine but not the influx of beta-alanine. However, the effect of PMA down-regulation and A23187 up......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...

  12. Tamoxifen induced multinucleated cells (symplasts) and distortion of seminiferous tubules in rat testis

    Institute of Scientific and Technical Information of China (English)

    UrbanJ.A.D'Souza

    2003-01-01

    Aim: To evaluate the effect of tamoxifen citrate on male reproductive system of rat. Methods: Groups of male rats were gavaged with tamoxifen at doses of 200 mg.kg·-1.d-1, 400 mg·kg·-1.d-1 or 800 mg.kg-1·d-1 in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5μm thickness, stained with H&E and analyzed microscopically. Results: There was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15. Conclusion: Tamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility. (Asian J Androl 2003 Sep; 5: 217-220)

  13. Physiological Functions and Regulation of the Na+/H+ Exchanger [NHE1] in Renal Tubule Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Patricia G Vallés

    2015-08-01

    Full Text Available The sodium-hydrogen exchanger isoform-1 [NHE1] is a ubiquitously expressed plasma membrane protein that plays a central role in intracellular pH and cell volume homeostasis by catalyzing an electroneutral exchange of extracellular sodium and intracellular hydrogen. Outside of this important physiological function, the NHE1 cytosolic tail domain acts as a molecular scaffold regulating cell survival and actin cytoskeleton organization through NHE1-dependent signaling proteins. NHE1 plays main roles in response to physiological stress conditions which in addition to cell shrinkage and acidification, include hypoxia and mechanical stimuli, such as cell stretch. NHE1-mediated modulation of programmed cell death results from the exchanger-mediated changes in pHi, cell volume, and/or [Na+]I; and, it has recently become known that regulation of cellular signaling pathways are involved as well. This review focuses on NHE1 functions and regulations. We describe evidence showing how these structural actions integrate with ion translocation in regulating renal tubule epithelial cell survival.

  14. Uremic Toxins Induce ET-1 Release by Human Proximal Tubule Cells, which Regulates Organic Cation Uptake Time-Dependently

    Directory of Open Access Journals (Sweden)

    Carolien M. S. Schophuizen

    2015-06-01

    Full Text Available In renal failure, the systemic accumulation of uremic waste products is strongly associated with the development of a chronic inflammatory state. Here, the effect of cationic uremic toxins on the release of inflammatory cytokines and endothelin-1 (ET-1 was investigated in conditionally immortalized proximal tubule epithelial cells (ciPTEC. Additionally, we examined the effects of ET-1 on the cellular uptake mediated by organic cation transporters (OCTs. Exposure of ciPTEC to cationic uremic toxins initiated production of the inflammatory cytokines IL-6 (117 ± 3%, p < 0.001, IL-8 (122 ± 3%, p < 0.001, and ET-1 (134 ± 5%, p < 0.001. This was accompanied by a down-regulation of OCT mediated 4-(4-(dimethylaminostyryl-N-methylpyridinium-iodide (ASP+ uptake in ciPTEC at 30 min (23 ± 4%, p < 0.001, which restored within 60 min of incubation. Exposure to ET-1 for 24 h increased the ASP+ uptake significantly (20 ± 5%, p < 0.001. These effects could be blocked by BQ-788, indicating activation of an ET-B-receptor-mediated signaling pathway. Downstream the receptor, iNOS inhibition by (N(G‐monomethyl‐l‐arginine l-NMMA acetate or aminoguanidine, as well as protein kinase C activation, ameliorated the short-term effects. These results indicate that uremia results in the release of cytokines and ET-1 from human proximal tubule cells, in vitro. Furthermore, ET-1 exposure was found to regulate proximal tubular OCT transport activity in a differential, time-dependent, fashion.

  15. Malpighian Tubule Cells in Overwintering Cave Crickets Troglophilus cavicola (Kollar, 1833 and T. neglectus Krauss, 1879 (Rhaphidophoridae, Ensifera.

    Directory of Open Access Journals (Sweden)

    Saška Lipovšek

    Full Text Available During winter, cave cricket larvae undergo dormancy in subterranean habitats; this dormancy is termed diapause in second year Troglophilus cavicola larvae because they mature during this time, and termed quiescence in T. neglectus, because they mature after dormancy. Here we used electron microscopy to analyze ultrastructural changes in the epithelial cells in the Malpighian tubules (MTs of T. cavicola during diapause, in order to compare them with previous findings on T. neglectus. Moreover, the autophagosomes were studied with immunofluorescence microscopy in both species. Although the basic ultrastructure of the cells was similar, specific differences appeared during overwintering. During this natural starvation period, the nucleus, rER, the Golgi apparatus and mitochondria did not show structural changes, and the spherites were exploited. The abundances of autophagic structures in both species increased during overwintering. At the beginning of overwintering, in both species and sexes, the rates of cells with autophagic structures (phagophores, autophagosomes, autolysosomes and residual bodies were low, while their rates increased gradually towards the end of overwintering. Between sexes, in T. cavicola significant differences were found in the autophagosome abundances in the middle and at the end, and in T. neglectus at the end of overwintering. Females showed higher rates of autophagic cells than males, and these were more abundant in T. cavicola. Thus, autophagic processes in the MT epithelial cells induced by starvation are mostly parallel in diapausing T. cavicola and quiescent T. neglectus, but more intensive in diapausing females.

  16. Malpighian Tubule Cells in Overwintering Cave Crickets Troglophilus cavicola (Kollar, 1833) and T. neglectus Krauss, 1879 (Rhaphidophoridae, Ensifera).

    Science.gov (United States)

    Lipovšek, Saška; Novak, Tone; Janžekovič, Franc; Weiland, Nina; Leitinger, Gerd

    2016-01-01

    During winter, cave cricket larvae undergo dormancy in subterranean habitats; this dormancy is termed diapause in second year Troglophilus cavicola larvae because they mature during this time, and termed quiescence in T. neglectus, because they mature after dormancy. Here we used electron microscopy to analyze ultrastructural changes in the epithelial cells in the Malpighian tubules (MTs) of T. cavicola during diapause, in order to compare them with previous findings on T. neglectus. Moreover, the autophagosomes were studied with immunofluorescence microscopy in both species. Although the basic ultrastructure of the cells was similar, specific differences appeared during overwintering. During this natural starvation period, the nucleus, rER, the Golgi apparatus and mitochondria did not show structural changes, and the spherites were exploited. The abundances of autophagic structures in both species increased during overwintering. At the beginning of overwintering, in both species and sexes, the rates of cells with autophagic structures (phagophores, autophagosomes, autolysosomes and residual bodies) were low, while their rates increased gradually towards the end of overwintering. Between sexes, in T. cavicola significant differences were found in the autophagosome abundances in the middle and at the end, and in T. neglectus at the end of overwintering. Females showed higher rates of autophagic cells than males, and these were more abundant in T. cavicola. Thus, autophagic processes in the MT epithelial cells induced by starvation are mostly parallel in diapausing T. cavicola and quiescent T. neglectus, but more intensive in diapausing females.

  17. ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer

    OpenAIRE

    Ajjimaporn Amornpan; Botsford Tom; Garrett Scott H; Sens Mary; Zhou Xu; Dunlevy Jane R; Sens Donald A; Somji Seema

    2012-01-01

    Abstract Background ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transform...

  18. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Sarró, Eduard, E-mail: eduard.sarro@vhir.org [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Jacobs-Cachá, Conxita, E-mail: conxita.jacobs@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Itarte, Emilio, E-mail: emili.itarte@uab.es [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Meseguer, Anna, E-mail: ana.meseguer@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain)

    2012-01-15

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC. Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also identified ER

  19. The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules

    DEFF Research Database (Denmark)

    Halberg, Kenneth Agerlin; Rainey, Stephanie M.; Veland, Iben Rønn

    2016-01-01

    Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most...... role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border....

  20. Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Wolff Natascha A

    2010-05-01

    Full Text Available Abstract Background The class 1 carcinogen cadmium (Cd2+ disrupts the E-cadherin/β-catenin complex of epithelial adherens junctions (AJs and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/β-catenin signaling are known to contribute to carcinogenesis. Results We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC. Cd2+ (25 μM, 3-9 h caused nuclear translocation of β-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of β-catenin with AJ components (E-cadherin, α-catenin and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1 were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. Conclusions Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

  1. Evaluation of “Dream Herb,” Calea zacatechichi, for Nephrotoxicity Using Human Kidney Proximal Tubule Cells

    Directory of Open Access Journals (Sweden)

    Miriam E. Mossoba

    2016-01-01

    Full Text Available A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. “Dream herb,” Calea zacatechichi, has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening of C. zacatechichi for safety especially important. We exposed human proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects of C. zacatechichi relative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support for the need to further scrutinize the safety of this herbal dietary supplement.

  2. Evaluation of “Dream Herb,” Calea zacatechichi, for Nephrotoxicity Using Human Kidney Proximal Tubule Cells

    Science.gov (United States)

    Flynn, Thomas J.; Vohra, Sanah; Wiesenfeld, Paddy; Sprando, Robert L.

    2016-01-01

    A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. “Dream herb,” Calea zacatechichi, has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening of C. zacatechichi for safety especially important. We exposed human proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects of C. zacatechichi relative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support for the need to further scrutinize the safety of this herbal dietary supplement. PMID:27703475

  3. Haptoglobin attenuates hemoglobin-induced heme oxygenase-1 in renal proximal tubule cells and kidneys of a mouse model of sickle cell disease.

    Science.gov (United States)

    Chintagari, Narendranath Reddy; Nguyen, Julia; Belcher, John D; Vercellotti, Gregory M; Alayash, Abdu I

    2015-03-01

    Sickle cell disease (SCD), a hereditary hemolytic disorder is characterized by chronic hemolysis, oxidative stress, vaso-occlusion and end-organ damage. Hemolysis releases toxic cell-free hemoglobin (Hb) into circulation. Under physiologic conditions, plasma Hb binds to haptoglobin (Hp) and forms Hb-Hp dimers. The dimers bind to CD163 receptors on macrophages for further internalization and degradation. However, in SCD patients plasma Hp is depleted and free Hb is cleared primarily by proximal tubules of kidneys. Excess free Hb in plasma predisposes patients to renal damage. We hypothesized that administration of exogenous Hp reduces Hb-mediated renal damage. To test this hypothesis, human renal proximal tubular cells (HK-2) were exposed to HbA (50μM heme) for 24h. HbA increased the expression of heme oxygenase-1 (HO-1), an enzyme which degrades heme, reduces heme-mediated oxidative toxicity, and confers cytoprotection. Similarly, infusion of HbA (32μM heme/kg) induced HO-1 expression in kidneys of SCD mice. Immunohistochemistry confirmed the increased HO-1 expression in the proximal tubules of the kidney. Exogenous Hp attenuated the HbA-induced HO-1 expression in vitro and in SCD mice. Our results suggest that Hb-mediated oxidative toxicity may contribute to renal damage in SCD and that Hp treatment reduces heme/iron toxicity in the kidneys following hemolysis.

  4. Damage of tubule cells in diabetic nephropathy type 2: Urinary N-acetyl-β-D-glucosaminidasis and γ-glutamil-transferasis

    Directory of Open Access Journals (Sweden)

    Vlatković Vlastimir

    2007-01-01

    Full Text Available Background/Aim. A damage of tubular epithelial cells is followed by the release of cell enzymes and production of proinflammatory compounds, which lead to the tubulointerstitial damage. The aim of this study was to examine the function of renal tubules in the patients with diabetes mellitus type 2 (DM type 2 and the various proteinuria degrees, to establish the damage of the proximal tubule cells caused by DM type 2 by determining urinary N-acetyl-β-D-glucosaminidasis (β-NAG and γ- glutamil-transferasis (γ-GT activity in urine, as well as to compare the obtained results in the examined groups of patients with the values in the healthy examinees. Methods. A complete examination of renal function and selective enzymuria was performed in 37 patients with DM type 2, and 14 healthy examinees as the controls. The patients were divided in three groups according to the degree of proteinuria. The first group consisted of the patients with diabetes without microalbuminuria; the second one consisted of the patients with proteinuria of < 300 mg/24 h, and microalbuminuria of >20 mg/24 h, while the third one included the patients with proteinuria of >300 mg/24 h. Results. In the patients with DM type 2 and the preserved global renal function, fractional excretion of sodium, potassium and phosphates, as well as renal threshold of phosphates concentration, were not sensitive parameters for discovering the damage of the renal tubule function. The determination of β-NAG activity proved to be the most sensitive parameter for early discovering of tubule cells damages. The difference among the examined groups was statistically highly significant. Conclusion. The increased presence of β-NAG in the urine of DM type 2 patients, pointed out an early tubular disorder and damage of cells, while γ-GT was a less sensitive indicator of this damage.

  5. Stem cells from glomerulus to distal tubule: a never-ending story?

    Directory of Open Access Journals (Sweden)

    Melania Puddu

    2016-08-01

    Full Text Available The growing interest of research in the field of renal stem cells and kidney regeneration aims to get results that allow its clinical application, favoring the birth and development of regenerative medicine.Nephrogenesis requires differentiation into epithelial cells of a population of progenitor mesenchymal cells. Since this process ends at 36-38 weeks of gestational age, it is quite likely to imagine that such a population disappears in the human kidney after birth. However, several studies have identified in different parts of the adult kidney cells having the characteristics of stem cells that would be involved in renal regenerative processes. They may be classified as resident mesenchymal/epithelial progenitors and often share the same genetic and epigenetic profile as progenitor stem cells active during embryonic life, thus suggesting a common origin.Current literature includes two lines of thought: one attributes to stem cells a fundamental role in renal regeneration processes while the other sustains the intervention of other mechanisms.The aim of this review is to report on progress made in research in the field of kidney regeneration starting from the past century and arriving at the present, with an analysis of scientific works that have produced the most important results in this field. Proceedings of the 2nd International Course on Perinatal Pathology (part of the 11th International Workshop on Neonatology · October 26th-31st, 2015 · Cagliari (Italy · October 31st, 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

  6. Single-nucleotide polymorphisms of the dopamine D2 receptor increase inflammation and fibrosis in human renal proximal tubule cells.

    Science.gov (United States)

    Jiang, Xiaoliang; Konkalmatt, Prasad; Yang, Yu; Gildea, John; Jones, John E; Cuevas, Santiago; Felder, Robin A; Jose, Pedro A; Armando, Ines

    2014-03-01

    The dopamine D2 receptor (D2R) negatively regulates inflammation in mouse renal proximal tubule cells (RPTCs), and lack or downregulation of the receptor in mice increases the vulnerability to renal inflammation independent of blood pressure. Some common single-nucleotide polymorphisms (SNPs; rs6276, rs6277, and rs1800497) in the human DRD2 gene are associated with decreased D2R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs (hRPTCs) expressing these SNPs have increased expression of inflammatory and injury markers. We studied immortalized hRPTCs carrying D2R SNPs and compared them with cells carrying no D2R SNPs. RPTCs with D2R SNPs had decreased D2R expression and function. The expressions of the proinflammatory tumor necrosis factor-α and the profibrotic transforming growth factor-β1 and its signaling targets Smad3 and Snail1 were increased in hRPTC with D2R SNPs. These cells also showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins, assessed by increased vimentin, fibronectin 1, and collagen I a1. To test the specificity of these D2R SNP effects, hRPTC with D2R SNPs were transfected with a plasmid encoding wild-type DRD2. The expression of D2R was increased and that of transforming growth factor-β1, Smad3, Snail1, vimentin, fibronectin 1, and collagen I a1 was decreased in hRPTC with D2R SNPs transfected with wild-type DRD2 compared with hRPTC-D2R SNP transfected with empty vector. These data support the hypothesis that D2R function has protective effects in hRPTCs and suggest that carriers of these SNPs may be prone to chronic renal disease and high blood pressure.

  7. Cdc42 regulates epithelial cell polarity and cytoskeletal function during kidney tubule development

    DEFF Research Database (Denmark)

    Elias, Bertha C; Das, Amrita; Parekh, Diptiben V

    2015-01-01

    The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development and main......The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development...... and maintenance, its exact molecular function in kidney development is not well understood. In this study, we define the specific role of Cdc42 during murine kidney epithelial tubulogenesis by deleting it selectively at the initiation of ureteric bud or metanephric mesenchyme development. Deletion in either...

  8. SOCS3 overexpression inhibits advanced glycation end product-induced EMT in proximal tubule epithelial cells.

    Science.gov (United States)

    Yu, Lin; Zhang, Ying; Zhang, Huimin; Li, Yingtao

    2017-06-01

    Diabetic nephropathy (DN) is among the most severe complications of diabetes mellitus, and may lead to end-stage renal disease. Sustained exposure to advanced glycation end products (AGEs) typically causes renal tubular epithelial cells (TECs) to suffer from an epithelial-to-mesenchymal transition (EMT). However, there remains no consensus regarding the mechanism underlying the cause of EMT in TECs as induced by AGEs. In the present study, we investigated the promotion of EMT in TECs by AGEs, and the activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. In addition, we constructed a recombinant adenovirus (Ad) that overexpressed suppressor of cytokine signaling 3 (SOCS3), and examined the regulatory role of SOCS3 in the activation of JAK/STAT signaling and the promotion of EMT in TECs. The results demonstrated that AGE-bovine serum albumin (BSA) treatment significantly promoted the expression of EMT-associated proteins, while reducing the expression of the epithelial cell marker, E-cadherin. Furthermore, the Ad-mediated SOCS3 overexpression markedly inhibited the AGE-BSA-induced JAK2/STAT3 activation; phosphorylated JAK2 and phosphorylated STAT3 expression levels were reduced by the Ad-SOCS3 infection, compared with the control Ad (Ad-con) infection, in HK-2 cells subject to AGE-BSA. Moreover, the overexpression of SOCS3 markedly inhibited the AGE-BSA-promoted EMT in HK-2 cells. AGE-BSA-promoted EMT-associated proteins, such as α-smooth muscle actin and collagen I, were reduced by the Ad-SOCS3 virus infection, in contrast to the Ad-con virus infection. Furthermore, reduced E-cadherin expression was reversed by the Ad-SOCS3 virus infection, in contrast to the Ad-con virus infection, in epithelial HK-2 cells. In conclusion, the present study confirmed the inhibitory role of SOCS3 in the AGE-induced EMT in renal TECs, implying the protective role of SOCS3 in DN.

  9. Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival.

    Science.gov (United States)

    Abu Jawdeh, Bassam G; Khan, Shenaz; Deschênes, Isabelle; Hoshi, Malcolm; Goel, Monu; Lock, Jeffrey T; Shinlapawittayatorn, Krekwit; Babcock, Gerald; Lakhe-Reddy, Sujata; DeCaro, Garren; Yadav, Satya P; Mohan, Maradumane L; Naga Prasad, Sathyamangla V; Schilling, William P; Ficker, Eckhard; Schelling, Jeffrey R

    2011-12-01

    Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

  10. Activation of the ALK-5 Pathway is not per se Sufficient for the Antiproliferative Effect of TGF-β1 on Renal Tubule Epithelial Cells

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    Omar García-Sánchez

    2015-10-01

    Full Text Available Background/Aims: Defective tissue repair underlies renal tissue degeneration during chronic kidney disease (CKD progression. Unbalanced presence of TGF-β opposes effective cell proliferation and differentiation processes, necessary to replace damaged epithelia. TGF-β also retains arrested cells in a fibrotic phenotype responsible for irreversible scarring. In order to identify prospective molecular targets to prevent the effect of TGF-β during CKD, we studied the signaling pathways responsible for the antiproliferative effect of this cytokine. Methods: Tubule epithelial HK2 and MDCK cells were treated with TGF-β (or not as control to study cell proliferation (by MTT, cell signaling (by Western blot, cell cycle (by flow cytometry and apoptosis (DNA fragmentation. Results: TGF-β fully activates the ALK-5 receptor pathway, whereas it has no effect on the ALK-1 and MAPK pathways in both HK2 and MDCK cells. Interestingly, TGF-β exerts an antiproliferative effect only on MDCK cells, through a cytostatic effect in G0/G1. Inhibition of the ALK-5 pathway with SB431542 prevents the cytostatic effect of TGF-β on MDCK cells. Conclusion: Activation of the ALK-5 pathway is not sufficient for the antiproliferative effect of TGF-β. The presence of undetermined permissive conditions or absence of undetermined inhibitory conditions seems to be necessary for this effect. The ALK-5 pathway appears to provide targets to modulate fibrosis, but further research is necessary to identify critical circumstances allowing or inhibiting its role at modulating tubule epithelial cell proliferation and tubule regeneration in the context of CKD progression.

  11. The effects of levamisole on urinary enzyme measurements and proximal tubule cell inclusions in male rats.

    Science.gov (United States)

    Evans, G. O.; Goodwin, D. A.; Parsons, C. E.; Read, N. G.

    1988-01-01

    A markedly increased incidence of large angular secondary lysosomes was observed in the proximal tubular cells of male Wistar rats dosed orally with levamisole, 75 mg/kg body weight for 15 days. These inclusions were similar in appearance to those previously observed in male rats treated with decahydronaphthalene. Urinary enzymes were measured throughout the study, and of these enzymes lactate dehydrogenase and N-acetyl-beta-D-glucosaminidase activities were higher on days 9 and 13 for rats dosed with levamisole in comparison with control animals. Urine volumes increased for the levamisole treatment group, but no treatment related changes of urine protein output were found. Images Figs. 1 & 2 Fig. 3 Fig. 4 PMID:2837266

  12. Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells.

    Science.gov (United States)

    Carranza, Andrea; Musolino, Patricia L; Villar, Marcelo; Nowicki, Susana

    2008-12-01

    The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.

  13. Morphometric evaluation of seminiferous tubule and proportionate numerical analysis of Sertoli and spermatogenic cells indicate differences between crossbred and purebred bulls

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    Utkarsh K. Tripathi

    2015-05-01

    Full Text Available Aim: The present study compared the testicular cytology and histology between crossbred (Holstein–Friesian [HF] × Tharparkar and purebred (HF and Tharparkar bulls to find out differences if any. Materials and Methods: Four peripubertal bulls from each breed were utilized for the study. Through percutaneous needle aspiration biopsy, Sertoli and spermatogenic cells were extracted, and morphometry was studied. For histological studies, testicular tissues obtained through unilateral castration were utilized. Sertoli cells specific GATA4 antibody was used to study the population of Sertoli cells in the seminiferous tubule through immunofluorescence. Results: The testicular weight, volume, and scrotal circumference differed significantly among the breeds. The diameter and area of the seminiferous tubule was high in HF, followed by Karan Fries (KF, and Tharparkar bulls. However, the degree of compactness, based on qualitative evaluation, was high in Tharparkar followed by KF and HF bulls. The intensity of Leydig cells was higher in Tharparkar bulls followed by KF and HF. The proportion of Sertoli cells was higher (p<0.05 in HF and Tharparkar bulls compared to KF bulls. Conclusion: It may be concluded that variations exist in testicular components of the breeds studied and the proportion of Sertoli cells in relation to spermatogenic cells was significantly lower in crossbred bulls compared to purebred bulls.

  14. Mechanisms of collective cell movement lacking a leading or free front edge in vivo.

    Science.gov (United States)

    Uechi, Hiroyuki; Kuranaga, Erina

    2017-08-01

    Collective cell movement is one of the strategies for achieving the complex shapes of tissues and organs. In this process, multiple cells within a group held together by cell-cell adhesion acquire mobility and move together in the same direction. In some well-studied models of collective cell movement, the mobility depends strongly on traction generated at the leading edge by cells located at the front. However, recent advances in live-imaging techniques have led to the discovery of other types of collective cell movement lacking a leading edge or even a free edge at the front, in a diverse array of morphological events, including tubule elongation, epithelial sheet extension, and tissue rotation. We herein review some of the developmental events that are organized by collective cell movement and attempt to elucidate the underlying cellular and molecular mechanisms, which include membrane protrusions, guidance cues, cell intercalation, and planer cell polarity, or chirality pathways.

  15. Epidermal growth factor decreases PEPT2 transport capacity and expression in the rat kidney proximal tubule cell line SKPT0193 cl.2

    DEFF Research Database (Denmark)

    Bravo, Silvina A; Nielsen, Carsten Uhd; Amstrup, Jan;

    2004-01-01

    transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined...... suggests that this might be disadvantageous when studying PEPT2-mediated transport phenomena. These findings demonstrate for the first time EGF-mediated regulation of PEPT2 expression in a kidney cell line. The relevance for kidney regulation of peptide transport activity in physiological and...... by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake...

  16. Proximal tubule epithelial cell specific ablation of the spermidine/spermine N1-acetyltransferase gene reduces the severity of renal ischemia/reperfusion injury.

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    Kamyar Zahedi

    Full Text Available BACKGROUND: Expression and activity of spermidine/spermine N1-acetyltransferase (SSAT increases in kidneys subjected to ischemia/reperfusion (I/R injury, while its ablation reduces the severity of such injuries. These results suggest that increased SSAT levels contribute to organ injury; however, the role of SSAT specifically expressed in proximal tubule epithelial cells, which are the primary targets of I/R injury, in the mediation of renal damage remains unresolved. METHODS: Severity of I/R injury in wt and renal proximal tubule specific SSAT-ko mice (PT-SSAT-Cko subjected to bilateral renal I/R injury was assessed using cellular and molecular biological approaches. RESULTS: Severity of the loss of kidney function and tubular damage are reduced in PT-SSAT-Cko- compared to wt-mice after I/R injury. In addition, animals treated with MDL72527, an inhibitor of polyamine oxidases, had less severe renal damage than their vehicle treated counter-parts. The renal expression of HMGB 1 and Toll like receptors (TLR 2 and 4 were also reduced in PT-SSAT-Cko- compared to wt mice after I/R injury. Furthermore, infiltration of neutrophils, as well as expression of tumor necrosis factor-α (TNF-α, monocyte chemoattractant protein-1 (MCP-1 and interleukin-6 (IL-6 transcripts were lower in the kidneys of PT-SSAT-Cko compared to wt mice after I/R injury. Finally, the activation of caspase3 was more pronounced in the wt compared to PT-SSAT-Cko animals. CONCLUSIONS: Enhanced SSAT expression by proximal tubule epithelial cells leads to tubular damage, and its deficiency reduces the severity of renal I/R injury through reduction of cellular damage and modulation of the innate immune response.

  17. Ochratoxin A activates opposing c-MET/PI3K/Akt and MAPK/ERK 1-2 pathways in human proximal tubule HK-2 cells.

    Science.gov (United States)

    Özcan, Zeynep; Gül, Gizem; Yaman, Ibrahim

    2015-08-01

    Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by filamentous fungi, such as Aspergillus and Penicillium. Because OTA is a common contaminant of food and feeds, humans and animals are frequently exposed to OTA in daily life. It has been classified as a carcinogen in rodents and a possible carcinogen in humans. OTA has been shown to deregulate a variety of different signal transduction pathways in a cell type- and dosage-depending manner resulting in contrasting physiological effects, such as survival or cell death. While the ERK1-2 and JNK/SAPK MAPK pathways are major targets, knowledge about their role in OTA-mediated cell survival and death is fragmented. Similarly, the contribution of the PI3K/Akt pathway to the carcinogenic effect of OTA in proximal tubule cells has not been elucidated in detail. In this study, we demonstrated that OTA induced sustained activation of the PI3K/Akt and MEK/ERK1-2 signaling pathways in a dose- and time-dependent manner in HK-2 cells. Chemical inhibition of ERK1-2 activation or overexpression of dominant-negative and kinase-dead MEK1 leads to increased cell viability and decreased apoptosis in OTA-treated cells. Blockage of PI3K/Akt with Wortmannin aggravated the negative effect of OTA on cell viability and increased the levels of apoptosis. Moreover, we identified the c-MET proto-oncogene as an upstream receptor tyrosine kinase responsible for OTA-induced activation of PI3K/Akt signaling in HK-2 cells. Our data suggest that OTA may potentiate carcinogenesis by sustained activation of c-MET/PI3K/Akt signaling through suppression of apoptosis induced by MEK/ERK1-2 activation in damaged renal proximal tubule epithelial cells.

  18. Hypoxia inducible factor 1-alpha (HIF-1 alpha is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

    Directory of Open Access Journals (Sweden)

    Elisa Conde

    Full Text Available Acute tubular necrosis (ATN caused by ischemia/reperfusion (I/R during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α, using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

  19. Transepithelial transport and metabolism of glycine in S1, S2, and S3 cell types of the rabbit proximal tubule.

    Science.gov (United States)

    Parks, Lisa D; Barfuss, Delon W

    2002-12-01

    In the first of two sets of experiments, the lumen-to-cell and cell-to-bath transport rates for glycine were measured in the isolated-perfused medullary pars recta (S3 cells) of the rabbit proximal tubule at multiple luminal glycine concentrations (0-2.0 mM). The lumen-to-cell transport of glycine was saturated, which permitted the calculation of the transport maximum of disappearance rate of glycine from the lumen (pmol.min(-1).mm tubular length(-1)), K(m) (mM), and paracellular leak (pmol.min(-1).mm tubular length(-1).mM(-1)) values for this transport mechanism; these values were 4.3, 0.3, and 0.03, respectively. The cell-to-bath transport did not saturate but showed a linear relationship to cellular glycine concentration, 0.58 pmol.min(-1).mm tubular length(-1).mM(-1). The second set of experiments characterized the transport rate, cellular accumulation, and metabolic rate of lumen-to-cell transported [(3)H]glycine in all segments (cell types) of the proximal tubule, pars convoluta (S1 cells), cortical pars recta (S2 cells), and medullary pars recta (S3 cells). These proximal tubular segments were isolated and perfused at a single glycine concentration of 11.2 microM. From the results of this study and previous work (Barfuss DW and Schafer JA. Am J Physiol 236: F149-F162, 1979), we conclude that the axial heterogeneity for glycine lumen-to-cell and cell-to-bath transport capacity extends to the medullary pars recta (S3 cells; S1 > S2 S3 for lumen-to-cell transport and S1 > S2 > S3 for cell-to-bath transport). Also, we conclude that lumen-to-cell transported glycine can be metabolized and its metabolic rate displays axial heterogeneity (S1 > S2 > S3). The physiological significances of these transport and metabolic characteristics of the S3 cell type permits the medullary pars recta to effectively recover glycine from very low luminal glycine concentrations and makes glycine available for protective and maintenance metabolism of the medullary pars recta.

  20. Tuning Collective Cell Migration by Cell-Cell Junction Regulation

    NARCIS (Netherlands)

    Friedl, P.; Mayor, R.

    2017-01-01

    Collective cell migration critically depends on cell-cell interactions coupled to a dynamic actin cytoskeleton. Important cell-cell adhesion receptor systems implicated in controlling collective movements include cadherins, immunoglobulin superfamily members (L1CAM, NCAM, ALCAM), Ephrin/Eph receptor

  1. Tuning Collective Cell Migration by Cell-Cell Junction Regulation

    NARCIS (Netherlands)

    Friedl, P.; Mayor, R.

    2017-01-01

    Collective cell migration critically depends on cell-cell interactions coupled to a dynamic actin cytoskeleton. Important cell-cell adhesion receptor systems implicated in controlling collective movements include cadherins, immunoglobulin superfamily members (L1CAM, NCAM, ALCAM), Ephrin/Eph

  2. The pro-oxidant gene p66shc increases nicotine exposure-induced lipotoxic oxidative stress in renal proximal tubule cells.

    Science.gov (United States)

    Arany, Istvan; Hall, Samuel; Reed, Dustin K; Dixit, Mehul

    2016-09-01

    Nicotine (NIC) exposure augments free fatty acid (FFA) deposition and oxidative stress, with a concomitant increase in the expression of the pro-oxidant p66shc. In addition, a decrease in the antioxidant manganese superoxide dismutase (MnSOD) has been observed in the kidneys of mice fed a high‑fat diet. The present study aimed to determine whether the pro‑oxidant p66shc mediates NIC‑dependent increases in renal oxidative stress by augmenting the production of reactive oxygen species (ROS) and suppressing the FFA‑induced antioxidant response in cultured NRK52E renal proximal tubule cells. Briefly, NRK52E renal proximal tubule cells were treated with 200 µM NIC, 100 µM oleic acid (OA), or a combination of NIC and OA. The expression levels of p66shc and MnSOD were modulated according to genetic methods. ROS production and cell injury, in the form of lactate dehydrogenase release, were subsequently detected. Promoter activity of p66shc and MnSOD, as well as forkhead box (FOXO)‑dependent transcription, was investigated using reporter luciferase assays. The results demonstrated that NIC exacerbated OA‑mediated intracellular ROS production and cell injury through the transcriptional activation of p66shc. NIC also suppressed OA‑mediated induction of the antioxidant MnSOD promoter activity through p66shc‑dependent inactivation of FOXO activity. Overexpression of p66shc and knockdown of MnSOD had the same effect as treatment with NIC on OA‑mediated lipotoxicity. These data may be used to generate a therapeutic means to ameliorate renal lipotoxicity in obese smokers.

  3. An angiotensin-(1-7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme.

    Science.gov (United States)

    Wilson, Bryan A; Cruz-Diaz, Nildris; Marshall, Allyson C; Pirro, Nancy T; Su, Yixin; Gwathmey, TanYa M; Rose, James C; Chappell, Mark C

    2015-03-15

    Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

  4. A new look at electrolyte transport in the distal tubule.

    Science.gov (United States)

    Eladari, Dominique; Chambrey, Régine; Peti-Peterdi, Janos

    2012-01-01

    The distal nephron plays a critical role in the renal control of homeostasis. Until very recently most studies focused on the control of Na(+), K(+), and water balance by principal cells of the collecting duct and the regulation of solute and water by hormones from the renin-angiotensin-aldosterone system and by antidiuretic hormone. However, recent studies have revealed the unexpected importance of renal intercalated cells, a subtype of cells present in the connecting tubule and collecting ducts. Such cells were thought initially to be involved exclusively in acid-base regulation. However, it is clear now that intercalated cells absorb NaCl and K(+) and hence may participate in the regulation of blood pressure and potassium balance. The second paradigm-challenging concept we highlight is the emerging importance of local paracrine factors that play a critical role in the renal control of water and electrolyte balance.

  5. Malformations of the epididymis, incomplete regression of the mesonephric tubules and hyperplasia of Leydig cells in canine persistence of Müllerian duct syndrome.

    Science.gov (United States)

    Whyte, Ana; Monteagudo, Luis V; Díaz-Otero, Angel; Lebrero, M Eugenia; Tejedor, M Teresa; Falceto, M Victoria; Whyte, Jaime; Gallego, Margarita

    2009-10-01

    Persistence of the Müllerian duct syndrome (PMDS) is a rare form of pseudohermaphroditism characterized by the presence of uterus and oviducts in otherwise normally differentiated SRY-positive 78 XY canine males. Undescended testicles are also common. We report a case of a male PMDS dog with a uterus and bilateral cryptorchidism. The dog had an incomplete regression of the mesonephric tubules. As a consequence of this an abnormally enlarged head of the epididymis was observed. In addition, an extreme reduction in size of both the body and the tail was found. Microscopic examination of both testicles revealed bilateral hyperplasia of Leydig cells. The progesterone blood level was measured by ELISA and was found to be abnormally high (3.18 ng/ml) compared to that of normal male dogs (lower than 1 ng/ml). Three months after surgical removal of the internal genitalia, the serum progesterone, testosterone and oestradiol levels were normal for a castrated male dog.

  6. A SILAC-Based Approach Elicits the Proteomic Responses to Vancomycin-Associated Nephrotoxicity in Human Proximal Tubule Epithelial HK-2 Cells.

    Science.gov (United States)

    Li, Zhi-Ling; Zhou, Shu-Feng

    2016-01-29

    Vancomycin, a widely used antibiotic, often induces nephrotoxicity, however, the molecular targets and underlying mechanisms of this side effect remain unclear. The present study aimed to examine molecular interactome and analyze the signaling pathways related to the vancomycin-induced nephrotoxicity in human proximal tubule epithelial HK-2 cells using the stable isotope labeling by amino acids in cell culture (SILAC) approach. The quantitative proteomic study revealed that there were at least 492 proteins interacting with vancomycin and there were 290 signaling pathways and cellular functions potentially regulated by vancomycin in HK-2 cells. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, EMT, and ROS generation. These findings suggest that vancomycin-induced proteomic responses in HK-2 cells involvefunctional proteins and pathways that regulate cell cycle, apoptosis, autophagy, and redox homeostasis. This is the first systemic study revealed the networks of signaling pathways and proteomic responses to vancomycin treatment in HK-2 cells, and the data may be used to discriminate the molecular and clinical subtypes and to identify new targets and biomarkers for vancomycin-induced nephrotoxic effect. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for drug-induced renal toxicity.

  7. The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish.

    Science.gov (United States)

    Zhang, Jiaojiao; Yuan, Shipeng; Vasilyev, Aleksandr; Amin Arnaout, M

    2015-11-01

    The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.

  8. A role for the circadian clock protein Per1 in the regulation of the NaCl co-transporter (NCC) and the with-no-lysine kinase (WNK) cascade in mouse distal convoluted tubule cells.

    Science.gov (United States)

    Richards, Jacob; Ko, Benjamin; All, Sean; Cheng, Kit-Yan; Hoover, Robert S; Gumz, Michelle L

    2014-04-25

    It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent (22)Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

  9. A Role for the Circadian Clock Protein Per1 in the Regulation of the NaCl Co-transporter (NCC) and the with-no-lysine Kinase (WNK) Cascade in Mouse Distal Convoluted Tubule Cells*

    Science.gov (United States)

    Richards, Jacob; Ko, Benjamin; All, Sean; Cheng, Kit-Yan; Hoover, Robert S.; Gumz, Michelle L.

    2014-01-01

    It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent 22Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4. PMID:24610784

  10. During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect.

    Directory of Open Access Journals (Sweden)

    Katarzyna Marchlewsk

    2008-04-01

    Full Text Available During establishment of spermatogenesis at the prepubertal age, an early germ cells apoptotic wave occurs likely aimed to remove abnormal germ cells and to maintain a proper cell number ratio between maturating germ cells and Sertoli cells. Here we assessed Sertoli and germ cell apoptosis in relation to morphological parameters of Sertoli cell maturation in neonatal rats under the influence of testosterone, estradiol and FSH given alone or in combinations. From postnatal day (PND 5th to 15th male rats were daily injected with: 1 2.5 mg of testosterone propionate (TP, or 2 12.5 microg of 17beta-estradiol benzoate (EB, or 3 TP+EB, or 4 7.5 IU of human purified FSH (hFSH, or 5 hFSH+EB or solvents (control-C. Autopsy was performed on PND 16th. Sertoli cell nuclei area and incidence of seminiferous tubule lumen formation (LF were taken as markers of Sertoli cell maturation. Sertoli and germ cell apoptosis was assessed using TUNEL method. In comparison with C, the area of Sertoli cell nuclei was significantly reduced after EB (25.7+/-2.0 vs. 30.9+/-1.6 microm2 for C, p<0.001 and increased after hFSH+EB (33.1+/-2.3 microm2, p<0.05. Incidence of LF was completely arrested by steroid hormone treatments given separately, significantly inhibited after TP+EB (median: 0.0%, vs. 2.0% for C p<0.05 and significantly enhanced after hFSH+EB (median: 51.0%, p<0.001. hFSH alone did not influence LF. Incidence of TUNEL positive Sertoli cells significantly increased after EB (median: 2.9% vs. 0.5% for C, p<0.05 or TP+EB (median: 2.2%, p<0.01 and was not affected by other treatments. Incidence of TUNEL positive germ cells increased significantly after EB alone (median: 4.4% vs. 2.5%, for C, p<0.01 and was significantly decreased by hFSH+EB (median: 0.5%, p<0.01. CONCLUSIONS: 1 Administration of testosterone or estradiol to immature rats inhibits Sertoli cell maturation. 2 Estradiol stimulates Sertoli and germ cell apoptosis while testosterone has no effect. 3

  11. Protein kinase A induces recruitment of active Na+,K+-ATPase units to the plasma membrane of rat proximal convoluted tubule cells

    Science.gov (United States)

    Carranza, Maria Luisa; Rousselot, Martine; Chibalin, Alexander V; Bertorello, Alejandro M; Favre, Hervé; Féraille, Eric

    1998-01-01

    The aim of this study was to investigate the mechanism of control of Na+,K+-ATPase activity by the cAMP-protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10−3 M dibutyryl-cAMP (db-cAMP) or 8-bromo-cAMP) and endogenous cAMP (direct activation of adenylyl cyclases by 10−5 M forskolin) on Na+,K+-ATPase activity and membrane trafficking.PKA activation stimulated both the cation transport and hydrolytic activity of Na+,K+-ATPase by about 40 %. Transport activity stimulation was specific to the PKA signalling pathway since (1) db-cAMP stimulated the ouabain-sensitive 86Rb+ uptake in a time- and dose-dependent fashion; (2) this effect was abolished by addition of H-89 or Rp-cAMPS, two structurally different PKA inhibitors; and (3) this stimulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db-cAMP on the hydrolytic activity of Na+,K+-ATPase was accounted for by an increased maximal ATPase rate (Vmax) without alteration of the efficiency of the pump, suggesting that cAMP-PKA pathway was implicated in membrane redistribution control.To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP-PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+-ATPase activity by db-cAMP was associated with an increase (+40 %) in Na+,K+-ATPase units expressed at the cell surface which was assessed by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (-30 %) in pump units located in the subcellular fraction corresponding to early endosomes.In conclusion, PKA stimulates Na+,K+-ATPase activity, at least in part, by increasing the number of

  12. Hepatitis B virus X protein promotes renal epithelial-mesenchymal transition in human renal proximal tubule epithelial cells through the activation of NF-κB.

    Science.gov (United States)

    Li, Mei; Hu, Liping; Zhu, Fengxin; Zhou, Zhangmei; Tian, Jianwei; Ai, Jun

    2016-08-01

    Hepatitis B virus (HBV)-associated glomerulo-nephritis is the most common extra-hepatic disorder occurring with hepatitis B virus infection. In the present study, we hypothesized that HBV X protein (HBx) may play a critical role in renal interstitial fibrosis, as HBx has been shown to induce epithelial-mesenchymal transition (EMT) in renal cells. For this purpose, we successfully transfected HBx plasmid into human renal proximal tubule epithelial cells (HK-2 cells). We found that transfection with HBx plasmid significantly downregulated E-cadherin expression and upregulated α-smooth muscle actin, collagen I and fibronectin expression in a time- and concentration-dependent manner (at the lower concentrations and earlier time points). HBx also increased nuclear factor-κB (NF-κB) phosphorylation in a time- and concentration-dependent manner (again at the lower concentrations and earlier time points); however, it did not alter the phosphorylation of Smad2, Smad3, p38, phosphoinositide 3-kinase (PI3K) or extracellular signal-regulated kinase (ERK). Thus, the findings of this study demonstrate that HBx promotes EMT in renal HK-2 cells, and the potential underlying mechanisms may involve the activation of the NF-κB signaling pathway.

  13. P2Y2 receptor activation inhibits the expression of the sodium-chloride cotransporter NCC in distal convoluted tubule cells.

    Science.gov (United States)

    Gailly, P; Szutkowska, M; Olinger, E; Debaix, H; Seghers, F; Janas, S; Vallon, V; Devuyst, O

    2014-11-01

    Luminal nucleotide stimulation is known to reduce Na(+) transport in the distal nephron. Previous studies suggest that this mechanism may involve the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which plays an essential role in NaCl reabsorption in the cells lining the distal convoluted tubule (DCT). Here we show that stimulation of mouse DCT (mDCT) cells with ATP or UTP promoted Ca(2+) transients and decreased the expression of NCC at both mRNA and protein levels. Specific siRNA-mediated silencing of P2Y2 receptors almost completely abolished ATP/UTP-induced Ca(2+) transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca(2+) concentration ([Ca(2+)]i) may control NCC transcription, we overexpressed the Ca(2+)-binding protein parvalbumin selectively in the cytosol or in the nucleus of mDCT cells. The decrease in NCC mRNA upon nucleotide stimulation was abolished in cells overexpressing cytosolic PV but not in cells overexpressing either a nuclear-targeted PV or a mutated PV unable to bind Ca(2+). Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from -1 to -2,200 bp was not regulated by changes in [Ca(2+)]i. In contrast, high cytosolic calcium level induced instability of NCC mRNA. We conclude that in mDCT cells: (1) P2Y2 receptor is essential for the intracellular Ca(2+) signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca(2+) concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA.

  14. Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

    Science.gov (United States)

    Féraille, Eric; Carranza, Maria Luisa; Gonin, Sandrine; Béguin, Pascal; Pedemonte, Carlos; Rousselot, Martine; Caverzasio, Joseph; Geering, Käthi; Martin, Pierre-Yves; Favre, Hervé

    1999-01-01

    Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT. PMID:10473631

  15. Sorting nexin 1 loss results in D5 dopamine receptor dysfunction in human renal proximal tubule cells and hypertension in mice.

    Science.gov (United States)

    Villar, Van Anthony M; Jones, John Edward; Armando, Ines; Asico, Laureano D; Escano, Crisanto S; Lee, Hewang; Wang, Xiaoyan; Yang, Yu; Pascua-Crusan, Annabelle M; Palmes-Saloma, Cynthia P; Felder, Robin A; Jose, Pedro A

    2013-01-04

    The peripheral dopaminergic system plays a crucial role in blood pressure regulation through its actions on renal hemodynamics and epithelial ion transport. The dopamine D5 receptor (D(5)R) interacts with sorting nexin 1 (SNX1), a protein involved in receptor retrieval from the trans-Golgi network. In this report, we elucidated the spatial, temporal, and functional significance of this interaction in human renal proximal tubule cells and HEK293 cells stably expressing human D(5)R and in mice. Silencing of SNX1 expression via RNAi resulted in the failure of D(5)R to internalize and bind GTP, blunting of the agonist-induced increase in cAMP production and decrease in sodium transport, and up-regulation of angiotensin II receptor expression, of which expression was previously shown to be negatively regulated by D(5)R. Moreover, siRNA-mediated depletion of renal SNX1 in C57BL/6J and BALB/cJ mice resulted in increased blood pressure and blunted natriuretic response to agonist in salt-loaded BALB/cJ mice. These data demonstrate a crucial role for SNX1 in D(5)R trafficking and that SNX1 depletion results in D(5)R dysfunction and thus may represent a novel mechanism for the pathogenesis of essential hypertension.

  16. Transforming growth factor-beta1 reduces megalin- and cubilin-mediated endocytosis of albumin in proximal-tubule-derived opossum kidney cells.

    Science.gov (United States)

    Gekle, Michael; Knaus, Petra; Nielsen, Rikke; Mildenberger, Sigrid; Freudinger, Ruth; Wohlfarth, Verena; Sauvant, Christoph; Christensen, Erik I

    2003-10-15

    Transforming growth factor (TGF)-beta1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial-mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF-beta1-induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin-cubilin-dependent receptor-mediated endocytosis. In this study we used a well established cell culture model (proximal-tubule-derived opossum kidney (OK) cells) in order to test the hypothesis that TGF-beta1 reduces megalin-cubilin-mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF-beta1 led to a time- and dose-dependent downregulation of megalin-cubilin-mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF-beta1 action does not involve mitogen-activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF-beta1-induced downregulation of megalin-cubilin-mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF-beta1. In conclusion our data indicate that enhanced levels of TGF-beta1 occurring in various nephropathies can lead to downregulation of megalin-cubilin-dependent endocytosis. Probably, TGF-beta1 leads to Smad2- and Smad3-dependent expression of negative regulators of receptor-mediated endocytosis.

  17. Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development.

    Science.gov (United States)

    Marra, Amanda N; Wingert, Rebecca A

    2016-03-15

    Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA

  18. Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane

    NARCIS (Netherlands)

    Schophuizen, Carolien M S; De Napoli, Ilaria E; Jansen, Jitske; Teixeira, Sandra; Wilmer, Martijn J; Hoenderop, Joost G J; Van den Heuvel, Lambert P W; Masereeuw, R.; Stamatialis, Dimitrios

    2015-01-01

    The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion

  19. Intracellular distribution, cell-to-cell trafficking and tubule-inducing activity of the 50 kDa movement protein of Apple chlorotic leaf spot virus fused to green fluorescent protein.

    Science.gov (United States)

    Satoh, H; Matsuda, H; Kawamura, T; Isogai, M; Yoshikawa, N; Takahashi, T

    2000-08-01

    The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP-GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP-GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287-457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.

  20. Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

    Science.gov (United States)

    de Michele, F; Poels, J; Weerens, L; Petit, C; Evrard, Z; Ambroise, J; Gruson, D; Wyns, C

    2017-01-01

    Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm(3) tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either

  1. Cell-specific expression of the glucocorticoid receptor within granular convoluted tubules of the rat submaxillary gland

    Energy Technology Data Exchange (ETDEWEB)

    Antakly, T.; Zhang, C.X.; Sarrieau, A.; Raquidan, D. (McGill Univ., Montreal, Quebec (Canada))

    1991-01-01

    The submaxillary gland, a heterogeneous tissue composed essentially of two functionally distinct cell types (tubular epithelial and acinar), offers an interesting system in which to study the mechanisms of steroid-dependent growth and differentiation. One cell type, the granular convoluted tubular (GCT) cell, secretes a large number of physiologically important polypeptides, including epidermal and nerve growth factors. Two steroids, androgens and glucocorticoids, greatly influence the growth, differentiation, and secretory activity of GCT cells. Because glucocorticoids can partially mimic or potentiate androgen effects, it has been thought that glucocorticoids act via androgen receptors. Since the presence of glucocorticoid receptors is a prerequisite for glucocorticoid action, we have investigated the presence and cellular distribution of glucocorticoid receptors within the rat submaxillary gland. Binding experiments using (3H)dexamethasone revealed the presence of high affinity binding sites in rat submaxillary tissue homogenates. Most of these sites were specifically competed by dexamethasone, corticosterone, and a pure glucocorticoid agonist RU 28362. Neither testosterone nor dihydrotestosterone competed for glucocorticoid binding. The cellular distribution of glucocorticoid receptors within the submaxillary gland was investigated by immunocytochemistry, using two highly specific glucocorticoid receptor antibodies. The receptor was localized in the GCT cells, but not in the acinar cells of rat and mouse submaxillary tissue sections. In GCT cells, the glucocorticoid receptor colocalized with several secretory polypeptides, including epidermal growth factor, nerve growth factor, alpha 2u-globulin, and atrial natriuretic factor.

  2. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  3. Fine structure of the malpighian tubule in Aedes aegypti

    Energy Technology Data Exchange (ETDEWEB)

    Mathew, G.; Rai, K.S.

    1976-07-01

    The malpighian tubule in Aedes aegypti adults is formed by 2 cell types: the principal cell which forms the great bulk of the tubule, and the stellate cell interspersed singly along the tubule. Both cell types possess ultrastructural features characteristic of cells engaged in ion balance and osmoregulation. These include extensive basal infolding and the differentiation of an apical brush border of microvilli. The central area of the cytoplasm of the principal cell is highly vacuolated while in the stellate cell it is finely granular lacking vacuoles. The microvilli in the principal cells enclose elongated, dense mitochondria whereas the stellate cell microvilli lack mitochondria. Excretory granules of an as yet unknown chemical nature accumulate in the principal cell cytoplasm after a blood meal.

  4. Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jason Bennett

    2016-04-01

    Full Text Available Epithelial-mesenchymal transition (EMT, a process which describes the trans-differentiation of epithelial cells into motile mesenchymal cells, is pivotal in stem cell behavior, development and wound healing, as well as contributing to disease processes including fibrosis and cancer progression. Maintenance immunosuppression with calcineurin inhibitors (CNIs has become routine management for renal transplant patient, but unfortunately the nephrotoxicity of these drugs has been well documented. HK-2 cells were exposed to Tacrolimus (FK506 and EMT markers were assessed by RT PCR and western blot. FK506 effects on TGF-β mRNA were assessed by RT PCR and TGF-β secretion was measured by ELISA. The impact of increased TGF-β secretion on Smad signaling pathways was investigated. The impact of inhibition of TGF-β signaling on EMT processes was assessed by scratch-wound assay. The results presented in this study suggest that FK506 initiates EMT processes in the HK-2 cell line, with altered expression of epithelial and myofibroblast markers evident. Additionally, the study demonstrates that FK506 activation of the TGF-β/ SMAD pathways is an essential step in the EMT process. Overall the results demonstrate that EMT is heavily involved in renal fibrosis associated with CNI nephrotoxicity.

  5. Potentiation by nitric oxide of cyclosporin A and FK506-induced apoptosis in renal proximal tubule cells.

    Science.gov (United States)

    Hortelano, S; Castilla, M; Torres, A M; Tejedor, A; Boscá, L

    2000-12-01

    Proximal tubular epithelial cells (PTEC) exhibit a high sensitivity to undergo apoptosis in response to proinflammatory stimuli and immunosuppressors and participate in the onset of several renal diseases. This study examined the expression of inducible nitric oxide (NO) synthase after challenge of PTEC with bacterial cell wall molecules and inflammatory cytokines and analyzed the pathways that lead to apoptosis in these cells by measuring changes in the mitochondrial transmembrane potential and caspase activation. The data show that the apoptotic effects of proinflammatory stimuli mainly were due to the expression of inducible NO synthase. Cyclosporin A and FK506 inhibited partially NO synthesis. However, both NO and immunosuppressors induced apoptosis, probably through a common mechanism that involved the irreversible opening of the mitochondrial permeability transition pore. Activation of caspases 3 and 7 was observed in cells treated with high doses of NO and with moderate concentrations of immunosuppressors. The conclusion is that the cooperation between NO and immunosuppressors that induce apoptosis in PTEC might contribute to the renal toxicity observed in the course of immunosuppressive therapy.

  6. Characterization of the Interaction of Staphylococcal Entertoxin B with CD1d Expressed in Human Renal Proximal Tubule Epithelial Cells

    Science.gov (United States)

    2015-02-04

    Software, Inc., San Diego , CA). RPTEC culture and fluorescence-based reporting RPTECs were grown in REBM culture medium supple- mented with a bullet kit...Med. 2007;232(9):1142 51. 32. Bendelac A, Rivera MN, Park SH, Roark JH. Mouse CD1 specific NK1 T cells: development, specificity, and function. Annu

  7. Smad mediated regulation of inhibitor of DNA binding 2 and its role in phenotypic maintenance of human renal proximal tubule epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mangalakumar Veerasamy

    Full Text Available The basic-Helix-Loop-Helix family (bHLH of transcriptional factors plays a major role in regulating cellular proliferation, differentiation and phenotype maintenance. The downregulation of one of the members of bHLH family protein, inhibitor of DNA binding 2 (Id2 has been shown to induce de-differentiation of epithelial cells. Opposing regulators of epithelial/mesenchymal phenotype in renal proximal tubule epithelial cells (PTEC, TGFβ1 and BMP7 also have counter-regulatory effects in models of renal fibrosis. We investigated the regulation of Id2 by these growth factors in human PTECs and its implication in the expression of markers of epithelial versus myofibroblastic phenotype. Cellular Id2 levels were reduced by TGFβ1 treatment; this was prevented by co-incubation with BMP7. BMP7 alone increased cellular levels of Id2. TGFβ1 and BMP7 regulated Id2 through Smad2/3 and Smad1/5 dependent mechanisms respectively. TGFβ1 mediated Id2 suppression was essential for α-SMA induction in PTECs. Although Id2 over-expression prevented α-SMA induction, it did not prevent E-cadherin loss under the influence of TGFβ1. This suggests that the loss of gate keeper function of E-cadherin alone may not necessarily result in complete EMT and further transcriptional re-programming is essential to attain mesenchymal phenotype. Although BMP7 abolished TGFβ1 mediated α-SMA expression by restoring Id2 levels, the loss of Id2 was not sufficient to induce α-SMA expression even in the context of reduced E-cadherin expression. Hence, a reduction in Id2 is critical for TGFβ1-induced α-SMA expression in this model of human PTECs but is not sufficient in it self to induce α-SMA even in the context of reduced E-cadherin.

  8. A novel role for c-Myc in G protein-coupled receptor kinase 4 (GRK4) transcriptional regulation in human kidney proximal tubule cells.

    Science.gov (United States)

    Gildea, John J; Tran, Hanh T; Van Sciver, Robert E; Bigler Wang, Dora; Carlson, Julia M; Felder, Robin A

    2013-05-01

    The G protein-coupled receptor kinase 4 (GRK4) negatively regulates the dopaminergic system by desensitizing the dopamine-1-receptor. The expressional control of GRK4 has not been reported, but here we show that the transcription factor c-Myc binds to the promoter of GRK4 and positively regulates GRK4 protein expression in human renal proximal tubule cells (RPTCs). Addition of phorbol esters to RPTCs not only increased c-Myc binding to the GRK4 promoter but also increased both phospho-c-Myc and GRK4 expression. The phorbol ester-mediated increase in GRK4 expression was completely blocked by the c-Myc inhibitor, 10074-G5, indicating that GRK4 is downstream of phospho-c-Myc. The autocrine production of angiotensin II (Ang II) in RPTCs increased the phosphorylation and activation of c-Myc and subsequently GRK4 expression. 3-Amino-4-thio-butyl sulfonate, an inhibitor of aminopeptidase A, increased RPTC secretion of Ang II. 3-Amino-4-thio-butyl sulfonate or Ang II increased the expression of both phospho-c-Myc and GRK4, which was blocked by 10074-G5. Blockade of the Ang II type 1 receptor with losartan decreased phospho-c-Myc and GRK4 expression. Both inhibition of c-Myc activity and blockade of Ang II type 1 receptor restored the coupling of dopamine-1-receptor to adenylyl cyclase stimulation in uncoupled RPTCs, whereas phorbol esters or Ang II caused the uncoupling of normally coupled RPTCs. We suggest that the Ang II type 1 receptor impairs dopamine-1-receptor function via c-Myc activation of GRK4. This novel pathway may be involved in the increase in blood pressure in hypertension that is mediated by increased activity of the renin-angiotensin system and decreased activity of the renal dopaminergic system.

  9. Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells.

    Science.gov (United States)

    Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae

    2012-04-01

    This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.

  10. Characterization of rPEPT2-mediated Gly-Sar transport parameters in the rat kidney proximal tubule cell line SKPT-0193 cl.2 cultured in basic growth media

    DEFF Research Database (Denmark)

    Bravo, Silvina A; Nielsen, Carsten Uhd; Frokjaer, Sven;

    2005-01-01

    added. The aim of the study was thus to characterize Gly-Sar transport parameters in SKPT cells cultured in basic growth media (conventional media without added agonists). Morphology was studied using confocal laser scanning microscopy (CLSM) and immunohistochemistry. Monolayer integrity was evaluated......The rat proximal kidney tubule cell line SKPT-0193 cl.2 (SKPT) expresses the di-/tripeptide transporter PEPT2 (rPEPT2) and has been used to study PEPT2-mediated transport. Traditionally, SKPT cells have been cultured in growth media supplemented with epidermal growth factor (EGF), apotransferrin...

  11. Determinants of leader cells in collective cell migration.

    NARCIS (Netherlands)

    Khalil, A.; Friedl, P.H.A.

    2010-01-01

    Collective migration is a basic mechanism of cell translocation during morphogenesis, wound repair and cancer invasion. Collective movement requires cells to retain cell-cell contacts, exhibit group polarization with defined front-rear asymmetry, and consequently move as one multicellular unit. Depe

  12. Emergence of oligarchy in collective cell migration

    Science.gov (United States)

    Schumacher, Linus; Maini, Philip; Baker, Ruth

    Identifying the principles of collective cell migration has the potential to help prevent birth defects, improve regenerative therapies and develop model systems for cancer metastasis. In collaboration with experimental biologists, we use computational simulations of a hybrid model, comprising individual-based stochastic cell movement coupled to a reaction-diffusion equation for a chemoattractant, to explore the role of cell specialisation in the guidance of collective cell migration. In the neural crest, an important migratory cell population in vertebrate embryo development, we present evidence that just a few cells are guiding group migration in a cell-induced chemoattractant gradient that determines the switch between ``leader'' and ``follower'' behaviour in individual cells. This leads us to more generally consider under what conditions cell specialisation might become advantageous for collective migration. Alternatively, individual cell responses to locally different microenvironmental conditions could create the (artefactual) appearance of heterogeneity in a population of otherwise identical cellular agents. We explore these questions using a self-propelled particle model as a minimal description for collective cell migration in two and three dimensions.

  13. Collective cell migration during inflammatory response

    Science.gov (United States)

    Wu, Di; Stroka, Kimberly; Aranda-Espinoza, Helim

    2012-02-01

    Wound scratch healing assays of endothelial cell monolayers is a simple model to study collective cell migration as a function of biological signals. A signal of particular interest is the immune response, which after initial wounding in vivo causes the release of various inflammatory factors such as tumor necrosis alpha (TNF-α). TNF-α is an innate inflammatory cytokine that can induce cell growth, cell necrosis, and change cell morphology. We studied the effects of TNF-α on collective cell migration using the wound healing assays and measured several migration metrics, such as rate of scratch closure, velocities of leading edge and bulk cells, closure index, and velocity correlation functions between migrating cells. We observed that TNF-α alters all migratory metrics as a function of the size of the scratch and TNF-α content. The changes observed in migration correlate with actin reorganization upon TNF-α exposure.

  14. Towards a Guided Regeneration of Renal Tubules at a Polyester Interstitium

    Directory of Open Access Journals (Sweden)

    Will W. Minuth

    2010-03-01

    Full Text Available Stem/progenitor cells are promising candidates for a therapy of renal failure. However, sound knowledge about implantation and regeneration is lacking. Therefore, mechanisms leading from stem/progenitor cells into tubules are under research. Renal stem/progenitor cells were isolated from neonatal rabbit kidney and mounted between layers of polyester fleece. It creates an artificial interstitium and replaces coating by extracellular matrix proteins. Tubulogenic development is induced by aldosterone. Electron microscopy illuminates growth of tubules in close vicinity to polyester fibers. Tubules contain a differentiated epithelium. The spatial extension of tubules opens a new strategy for testing morphogenic drugs and biocompatible fleece materials.

  15. Effect of BSA-induced ER stress on SGLT protein expression levels and alpha-MG uptake in renal proximal tubule cells.

    Science.gov (United States)

    Lee, Yu Jin; Suh, Han Na; Han, Ho Jae

    2009-06-01

    Recent studies demonstrated that endoplasmic reticulum (ER) stress regulates glucose homeostasis and that ER stress preconditioning which induces an adaptive, protective unfolded protein response (UPR) offers cytoprotection against nephrotoxins. Thus the aim of the present study was to use renal proximal tubule cells (PTCs) to further elucidate the link between the BSA-induced ER stress and alpha-methyl-d-glucopyranoside (alpha-MG) uptake and to identify related signaling pathways. Among ER stress inducers such as high glucose, BSA, H2O2, or tumicamycin, BSA pretreatment ameliorated the reduction of Na(+)-glucose cotransporter (SGLT) expression and alpha-MG uptake by gentamicin or cyclosporine A. Immunofluorescence studies revealed that BSA (10 mg/ml) stimulated the expression of glucose-regulated protein 78 (GRP78), an ER stress biomarker. In addition, BSA increased levels of GRP78 protein expression and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation in a time-dependent manner. Furthermore, transfection with a GRP78-specific small interfering RNA (siRNA) inhibited BSA-stimulated SGLT expression and alpha-MG uptake. In experiments designed to unravel the mechanisms underlying BSA-induced ER stress, BSA stimulated the production of cellular reactive oxygen species (ROS), and antioxidants such as ascorbic acid or N-acetylcysteine (NAC) blocked BSA-induced increases in GRP78 activation, eIF2alpha phosphorylation, SGLT expression, and alpha-MG uptake. Moreover, the cells upregulated peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA levels in response to BSA or troglitazone (a PPARgamma agonist), but BSA was ineffective in the presence of GW9662 (a PPARgamma antagonist). In addition, both BSA and troglitazone stimulated GRP78 and eIF2alpha activation, SGLT expression, and alpha-MG uptake, whereas GW9662 inhibited the effects of BSA. BSA also stimulated phosphorylation of JNK and NF-kappaB, and GW9662 or GRP78 siRNA attenuated this

  16. Mouse amnionless, which is required for primitive streak assembly, mediates cell-surface localization and endocytic function of cubilin on visceral endoderm and kidney proximal tubules.

    Science.gov (United States)

    Strope, Sharon; Rivi, Roberta; Metzger, Thomas; Manova, Katia; Lacy, Elizabeth

    2004-10-01

    Impaired primitive streak assembly in the mouse amnionless (amn) mutant results in the absence of non-axial trunk mesoderm, a derivative of the middle region of the primitive streak. In addition, the epiblast of amn mutants fails to increase significantly in size after E7.0, indicating that middle primitive streak assembly is mechanistically tied to the growth of the embryo during gastrulation. Amn, a novel transmembrane protein, is expressed exclusively in an extra-embryonic tissue, visceral endoderm (VE), during the early post-implantation stages. We show that Amn is also expressed in kidney proximal tubules (KPT) and intestinal epithelium, which, like the VE, are polarized epithelia specialized for resorption and secretion. To explore whether Amn participates in the development or function of KPT and intestinal epithelia and to gain insight into the function of Amn during gastrulation, we constructed Amn(-/-) ES cell+/+ blastocyst chimeras. While chimeras form anatomically normal kidneys and intestine, they exhibit variable, selective proteinuria, a sign of KPT malfunction. In humans, AMN has been genetically connected to Cubilin (CUBN), a multi-ligand scavenger receptor expressed by KPT, intestine and yolk sac. Loss of CUBN, the intestinal intrinsic factor (IF)-vitamin B12 receptor, results in hereditary megaloblastic anemia (MGA1), owing to vitamin B12 malabsorption. The recent report of MGA1 families with mutations in AMN suggests that AMN functions in the same pathway as CUBN. We demonstrate that Cubn is not properly localized to the cell surface in Amn(-/-) tissues in the embryo and adult mouse, and that adult chimeras exhibit selective proteinuria of Cubn ligands. This study demonstrates that Amn is an essential component of the Cubn receptor complex in vivo and suggests that Amn/Cubn is required for endocytosis/transcytosis of one or more ligands in the VE during gastrulation to coordinate growth and patterning of the embryo. Furthermore, as AMN is

  17. Glucosamine-induced Sp1 O-GlcNAcylation ameliorates hypoxia-induced SGLT dysfunction in primary cultured renal proximal tubule cells.

    Science.gov (United States)

    Suh, Han Na; Lee, Yu Jin; Kim, Mi Ok; Ryu, Jung Min; Han, Ho Jae

    2014-10-01

    The aim of this study is to determine whether GlcN could recover the endoplasmic reticulum (ER) stress-induced dysfunction of Na(+) /glucose cotransporter (SGLT) in renal proximal tubule cells (PTCs) under hypoxia. With the rabbit model, the renal ischemia induced tubulointerstitial abnormalities and decreased SGLTs expression in tubular brush-border, which were recovered by GlcN. Thus, the protective mechanism of GlcN against renal ischemia was being examined by using PTCs. Hypoxia decreased the level of protein O-GlcNAc and the expression of O-GlcNAc transferase (OGT) while increased O-GlcNAcase (OGA) and these were reversed by GlcN. Hypoxia also decreased the expression of SGLTs (SGLT1 and 2) and [(14) C]-α-methyl-D-glucopyranoside (α-MG) uptake which were recovered by GlcN and PUGNAc (OGA inhibitor). Hypoxia enhanced reactive oxygen species (ROS) and then ER stress proteins, glucose-regulated protein 78 (GRP78), and C/EBP-homologous protein (CHOP). However, the expression of GRP78 increased till 6 h and then decreased whereas CHOP increased gradually. Moreover, decreased GRP78 and increased CHOP were reversed by NAC (antioxidant) and GlcN. GlcN ameliorated hypoxia-induced decrease of O-GlcNAc modification of Sp1 but OGT or Sp1 siRNAs blocked the recovery effect of GlcN on SGLT expression and α-MG uptake. In addition, hypoxia-decreased GRP78 and HIF-1α expression was reversed by GlcN but OGT siRNA or Sp1 siRNA ameliorated the effect of GlcN. When PTCs were transfected with GRP78 siRNA or HIF-1α siRNA, SGLT expression and α-MG uptake was decreased. Taken together, these data suggest that GlcN-induced O-GlcNAc modified Sp1 with stimulating GRP78 and HIF-1α activity ameliorate hypoxia-induced SGLT dysfunction in renal PTCs. J. Cell. Physiol. 229: 1557-1568, 2014. © 2014 Wiley Periodicals, Inc.

  18. Bursts of activity in collective cell migration

    CERN Document Server

    Chepizhko, Oleksandr; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stephane; Alava, Mikko J; Zapperi, Stefano; La Porta, Caterina A M

    2016-01-01

    Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems.

  19. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    Energy Technology Data Exchange (ETDEWEB)

    Carette, Diane [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Perrard, Marie-Hélène, E-mail: marie-helene.durand@ens-lyon.fr [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Prisant, Nadia [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Gilleron, Jérome; Pointis, Georges [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); Segretain, Dominique [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Kallistem SAS Ecole Normale Supérieure de Lyon, Lyon (France)

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  20. Cutting through the complexity of cell collectives.

    Science.gov (United States)

    Nadell, Carey D; Bucci, Vanni; Drescher, Knut; Levin, Simon A; Bassler, Bonnie L; Xavier, João B

    2013-03-22

    Via strength in numbers, groups of cells can influence their environments in ways that individual cells cannot. Large-scale structural patterns and collective functions underpinning virulence, tumour growth and bacterial biofilm formation are emergent properties of coupled physical and biological processes within cell groups. Owing to the abundance of factors influencing cell group behaviour, deriving general principles about them is a daunting challenge. We argue that combining mechanistic theory with theoretical ecology and evolution provides a key strategy for clarifying how cell groups form, how they change in composition over time, and how they interact with their environments. Here, we review concepts that are critical for dissecting the complexity of cell collectives, including dimensionless parameter groups, individual-based modelling and evolutionary theory. We then use this hybrid modelling approach to provide an example analysis of the evolution of cooperative enzyme secretion in bacterial biofilms.

  1. Estradiol-17beta-BSA stimulates Ca(2+) uptake through nongenomic pathways in primary rabbit kidney proximal tubule cells: involvement of cAMP and PKC.

    Science.gov (United States)

    Han, H J; Lee, Y H; Park, S H

    2000-04-01

    The effect of estradiol-17beta-BSA (E(2)-BSA) on Ca(2+) uptake and its related signal pathways were examined in the primary cultured rabbit kidney proximal tubule cells. E(2)-BSA (10(-9) M) significantly stimulated Ca(2+) uptake from 2 h by 13% and at 8 h by 35% as compared to control, respectively. This stimulatory effect of E(2)-BSA was not inhibited by tamoxifen (10(-8) M, an intracellular estrogen receptor antagonist), actinomycin D (10(-7) M, a transcription inhibitor), and cycloheximide (4 x 10(-5) M, a protein synthesis inhibitor). However, E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by methoxyverapamil (10(-6) M, an L-type calcium channel blocker) and 5-(N-ethyl-N-isopropyl)-amiloride (10(-5) M, a Na(+)/H(+) antiporter blocker). These results suggest that E(2)-BSA stimulates Ca(2+) uptake through nongenomic pathways. Thus, we investigated which signal pathways were related to E(2)-BSA-induced stimulation of Ca(2+) uptake. 8-Br-cAMP (10(-6) M) alone increased Ca(2+) uptake by 22% compared to control. When E(2)-BSA combined with 8-Br-cAMP, Ca(2+) uptake was not significantly stimulated compared to E(2)-BSA. SQ 22536 (10(-6) M, an adenylate cyclase inhibitor) and myristoylated protein kinase A inhibitor amide 14-22 (10(-6) M, a protein kinase A inhibitor) blocked E(2)-BSA-induced stimulation of Ca(2+) uptake and E(2)-BSA also increased cAMP generation by 26% of that of control. In addition, TPA (0.02 ng/ml, an artificial PKC promoter) stimulated the Ca(2+) uptake by 14%, and the cotreatment of TPA and E(2)-BSA did not significantly stimulate Ca(2+) uptake compared to E(2)-BSA. E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by U 73122 (10(-6) M, a phospholipase C inhibitor) or bisindolylmaleimide I (10(-6) M, a protein kinase C inhibitor). Indeed, E(2)-BSA stimulated PKC activity by 26%. In conclusion, E(2)-BSA (10(-9) M) stimulated Ca(2+) uptake by nongenomic action, which is mediated by cAMP and PKC pathways.

  2. Administration of testosterone inhibits initiation of seminal tubule growth and decreases Sertoli cell number in the earliest period of rat's postnatal development.

    Directory of Open Access Journals (Sweden)

    Elzbieta Oszukowska

    2010-01-01

    Full Text Available Sertoli cell (SC number determines testes size and their capacity to produce spermatozoa. In the rat SC proliferate until 15th postnatal day (PND. Their proliferation is stimulated by FSH and inhibited by estradiol, but the role for androgens is uncertain. In this study we analyzed the effects of testosterone administration on testes growth and SC number in relation to timing of the treatment. Male rats were injected with 2.5 mg of testosterone propionate (TP from birth until 5th PND and autopsied either on 6th PND [TP1-5(6] or on 16th PND [TP1-5(16] (transient administration. Other rats received TP from birth until 15th PND [TP1-15] or between 5th and 15th PND [TP5-15] continuously and were autopsied on day 16th. Control groups (C received vehicle. In the Cs serum level of estradiol was 20-fold higher (p<0.001 and FSH was 1,7-fold higher (p<0.05 on 6th PND than on 16th PND, while testosterone did not change. After TP blood level of testosterone increased 2200-fold on 6th PND (p<0.05, and 8-fold on 16th PND. In turn, continuous TP administrations resulted on 16th PND in the increase in testosterone serum level by 2000-times of C without influence on FSH. While the treatment from birth either during initial 5 days or continuously until 15th day decreased testicular weight (p<0.001, tubule length (p<0.05 and SC number (p<0.001, the treatment initiated on 5th PND had no effects. TP reduced serum estradiol level on 6th PND by 13-fold (p<0.01, but doubled it on 16th PND. Conclusion: Neonatal rats secrete estradiol and FSH in the amounts greatly extending those presented during further development. Testosterone inhibits testicular growth and SC number acting during first 5 neonatal days by decreasing FSH secretion, but is not effective during further development. Direct inhibitory influence of testosterone or trough its increased aromatisation to estradiol beyond neonatal period may be responsible for sustained inhibition of testes growth and SC

  3. Entropy measures of collective cell migration

    Science.gov (United States)

    Whitby, Ariadne; Parrinello, Simona; Faisal, Aldo

    2015-03-01

    Collective cell migration is a critical process during tissue formation and repair. To this end there is a need to develop tools to quantitatively measure the dynamics of collective cell migration obtained from microscopy data. Drawing on statistical physics we use entropy of velocity fields derived from dense optic flow to quantitatively measure collective migration. Using peripheral nerve repair after injury as experimental system, we study how Schwann cells, guided by fibroblasts, migrate in cord-like structures across the cut, paving a highway for neurons. This process of emergence of organised behaviour is key for successful repair, yet the emergence of leader cells and transition from a random to ordered state is not understood. We find fibroblasts induce correlated directionality in migrating Schwann cells as measured by a decrease in the entropy of motion vector. We show our method is robust with respect to image resolution in time and space, giving a principled assessment of how various molecular mechanisms affect macroscopic features of collective cell migration. Finally, the generality of our method allows us to process both simulated cell movement and microscopic data, enabling principled fitting and comparison of in silico to in vitro. ICCS, Imperial College London & MRC Clinical Sciences Centre.

  4. Modeling traction forces in collective cell migration

    Science.gov (United States)

    Zimmermann, Juliane; Basan, Markus; Hayes, Ryan L.; Rappel, Wouter-Jan; Levine, Herbert

    2015-03-01

    Collective cell migration is an important process in embryonic development, wound healing, and cancer metastasis. We have developed a particle-based simulation for collective cell migration that describes flow patterns and finger formation at the tissue edge observed in wound healing experiments. We can apply methods for calculating intercellular stress to our simulation model, and have thereby provided evidence for the validity of a stress reconstitution method from traction forces used in experiments. To accurately capture experimentally measured traction forces and stresses in the tissue, which are mostly tensile, we have to include intracellular acto-myosin contraction into our simulation. We can then reproduce the experimentally observed behavior of cells moving around a circular obstacle, and suggest underlying mechanisms for cell-cell alignment and generation of traction force patterns.

  5. Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro

    OpenAIRE

    Kolman, Pavel; Pica, Angelo; Carvou, Nicolas; Boyde, Alan; Cockcroft, Shamshad; Loesch, Andrew; Pizzey, Arnold; Simeoni, Mariadelina; Capasso, Giovambattista; Unwin, Robert J.

    2009-01-01

    We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules;...

  6. Physical models of collective cell motility: from cell to tissue

    Science.gov (United States)

    Camley, B. A.; Rappel, W.-J.

    2017-03-01

    In this article, we review physics-based models of collective cell motility. We discuss a range of techniques at different scales, ranging from models that represent cells as simple self-propelled particles to phase field models that can represent a cell’s shape and dynamics in great detail. We also extensively review the ways in which cells within a tissue choose their direction, the statistics of cell motion, and some simple examples of how cell–cell signaling can interact with collective cell motility. This review also covers in more detail selected recent works on collective cell motion of small numbers of cells on micropatterns, in wound healing, and the chemotaxis of clusters of cells.

  7. Collective cell migration: a mechanistic perspective.

    Science.gov (United States)

    Vedula, Sri Ram Krishna; Ravasio, Andrea; Lim, Chwee Teck; Ladoux, Benoit

    2013-11-01

    Collective cell migration is fundamental to gaining insights into various important biological processes such as wound healing and cancer metastasis. In particular, recent in vitro studies and in silico simulations suggest that mechanics can explain the social behavior of multicellular clusters to a large extent with minimal knowledge of various cellular signaling pathways. These results suggest that a mechanistic perspective is necessary for a comprehensive and holistic understanding of collective cell migration, and this review aims to provide a broad overview of such a perspective.

  8. Collective cell migration: a physics perspective

    Science.gov (United States)

    Hakim, Vincent; Silberzan, Pascal

    2017-07-01

    Cells have traditionally been viewed either as independently moving entities or as somewhat static parts of tissues. However, it is now clear that in many cases, multiple cells coordinate their motions and move as collective entities. Well-studied examples comprise development events, as well as physiological and pathological situations. Different ex vivo model systems have also been investigated. Several recent advances have taken place at the interface between biology and physics, and have benefitted from progress in imaging and microscopy, from the use of microfabrication techniques, as well as from the introduction of quantitative tools and models. We review these interesting developments in quantitative cell biology that also provide rich examples of collective out-of-equilibrium motion.

  9. From cyst to tubule: innovations in vertebrate spermatogenesis.

    Science.gov (United States)

    Yoshida, Shosei

    2016-01-01

    Although vertebrates share many common traits, their germline development and function exhibit significant divergence. In particular, this article focuses on their spermatogenesis. The fundamental elements that constitute vertebrate spermatogenesis and the evolutionary changes that occurred upon transition from water to land will be discussed. The life-long continuity of spermatogenesis is supported by the function of stem cells. Series of mitotic and meiotic germ cell divisions are 'incomplete' due to incomplete cytokinesis, forming syncytia interconnected via intercellular bridges (ICBs). Throughout this process, germ cells are supported by appropriate microenvironments established primarily by somatic Sertoli cells. In anamniotes (fish and amphibians) spermatogenesis progresses in cysts, in which developing germ cell syncytia are individually encapsulated by Sertoli cells. Accordingly, Sertoli cells undergo turnover with germ cells that they nourish. This mode of cystic spermatogenesis is also observed in nonvertebrates as insects. In amniotes (reptiles, birds, and mammals), however, Sertoli cells do not turn over but comprise a persistent structure of seminiferous tubules. Sertoli cells nourish different stages of germ cells simultaneously in distinct regions of their surface. This function of Sertoli cells is spatiotemporally orchestrated, and the seminiferous epithelial cycle and spermatogenic wave make the seminiferous tubules a high-throughput factory for sperm production. Furthermore, contrary to the organized differentiating cells, undifferentiated spermatogonia that comprise the stem cell compartment exhibit active motion over the basal layer of seminiferous tubules and the frequent breakdown of ICBs. Thus, amniote seminiferous tubules represent a typical facultative (or open) niche environment without a stem cell tethering anatomically defined niche. WIREs Dev Biol 2016, 5:119-131. doi: 10.1002/wdev.204 For further resources related to this article

  10. Isolation of surface tubules of fowlpox virus.

    Science.gov (United States)

    Carter, J K; Cheville, N F

    1981-01-01

    Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.

  11. Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of Drosophila melanogaster

    Indian Academy of Sciences (India)

    Madhu G Tapadia; Naveen K Gautam

    2011-08-01

    Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of -tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.

  12. Collective dynamics of cell migration and cell rearrangements

    Science.gov (United States)

    Kabla, Alexandre

    Understanding multicellular processes such as embryo development or cancer metastasis requires to decipher the contributions of local cell autonomous behaviours and long range interactions with the tissue environment. A key question in this context concerns the emergence of large scale coordination in cell behaviours, a requirement for collective cell migration or convergent extension. I will present a few examples where physical and mechanical aspects play a significant role in driving tissue scale dynamics. Geometrical confinement is one of the key external factors influencing large scale coordination during collective migration. Using a combination of in vitro experiments and numerical simulations, we show that the velocity correlation length, measured in unconfined conditions, provides a convenient length scale to predict the dynamic response under confinement. The same length scale can also be used to quantify the influence range of directional cues within the cell population. Heterogeneity within motile cell populations is frequently associated with an increase in their invasive capability and appears to play an important role during cancer metastasis. Using in silico experiments, we studied the way cell invasion is influenced by both the degree of cell coordination and the amount of variability in the motile force of the invading cells. Results suggest that mechanical heterogeneity dramatically enhances the invasion rate through an emerging cooperative process between the stronger and weaker cells, accounting for a number of observed invasion phenotypes. Effective convergent extension requires on a consistent orientation of cell intercalation at the tissue scale, most often in relation with planar cell polarity mechanisms to define the primary axes of deformation. Using a novel modelling approach for cells mechanical interactions, we studied the dynamics of substrate free motile cell populations. Ongoing work shows in particular that nematic order emerges

  13. Collective Cell Movement Promotes Synchronization of Coupled Genetic Oscillators

    OpenAIRE

    Uriu, Koichiro; Morelli, Luis G.

    2014-01-01

    Collective cell movement is a crucial component of embryonic development. Intercellular interactions regulate collective cell movement by allowing cells to transfer information. A key question is how collective cell movement itself influences information flow produced in tissues by intercellular interactions. Here, we study the effect of collective cell movement on the synchronization of locally coupled genetic oscillators. This study is motivated by the segmentation clock in zebrafish somito...

  14. Natural history of seminiferous tubule degeneration in Klinefelter syndrome.

    Science.gov (United States)

    Aksglaede, Lise; Wikström, Anne M; Rajpert-De Meyts, Ewa; Dunkel, Leo; Skakkebaek, Niels E; Juul, Anders

    2006-01-01

    Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates dramatically at the time of puberty with complete hyalinization of the seminiferous tubules, although a few tubules with spermatogenesis may be present in adult life. Activation of the pituitary-gonadal axis at 3 months of age is seen in Klinefelter boys similar to healthy boys. However, the level of testosterone in Klinefelter boys is significantly lower than in controls. After this 'minipuberty', the hormone levels decline to normal prepubertal levels until puberty. In puberty, an initial rise in testosterone, inhibin B, LH and FSH occurs in Klinefelter boys. However, the rise in testosterone levels off and ends at a low-normal level in young adults. Likewise, serum concentration of inhibin B exhibits a dramatic decline to a low, often undetectable level, concomitantly with a rise in FSH, reflecting the degeneration of the seminiferous tubules. Many hypotheses about the underlying mechanism of the depletion of the germ cells in Klinefelter males have been reported and include insufficient supranumerary X-chromosome inactivation, Leydig cell insufficiency and disturbed regulation of apoptosis of Sertoli and Leydig cells. However, at present, the exact mechanism remains unclear. In this article, we summarize current knowledge on the development of the classical endocrinological and histological features of 47,XXY males from fetus to adulthood and review the literature concerning the degeneration of the seminiferous tubules in this syndrome.

  15. Membrane tubulation by elongated and patchy nanoparticles

    CERN Document Server

    Raatz, Michael

    2016-01-01

    Advances in nanotechnology lead to an increasing interest in how nanoparticles interact with biomembranes. Nanoparticles are wrapped spontaneously by biomembranes if the adhesive interactions between the particles and membranes compensate for the cost of membrane bending. In the last years, the cooperative wrapping of spherical nanoparticles in membrane tubules has been observed in experiments and simulations. For spherical nanoparticles, the stability of the particle-filled membrane tubules strongly depends on the range of the adhesive particle-membrane interactions. In this article, we show via modeling and energy minimization that elongated and patchy particles are wrapped cooperatively in membrane tubules that are highly stable for all ranges of the particle-membrane interactions, compared to individual wrapping of the particles. The cooperative wrapping of linear chains of elongated or patchy particles in membrane tubules may thus provide an efficient route to induce membrane tubulation, or to store such...

  16. CFTR mediated chloride secretion in the avian renal proximal tubule.

    Science.gov (United States)

    Laverty, Gary; Anttila, Ashley; Carty, Jenava; Reddy, Varudhini; Yum, Jamie; Arnason, Sighvatur S

    2012-01-01

    In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl(-)-dependent short circuit current (I(SC)) response, consistent with net transepithelial Cl(-) secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl(-) secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I(SC) responses in filter grown cultured monolayers. Bumetanide (10 μM), a specific blocker of NKCC, inhibited forskolin activated I(SC) by about 40%, suggesting that basolateral uptake of Cl(-) is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl(-) conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl(-) gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl(-) current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 μM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl(-) channel to mediate cAMP-activated Cl(-) secretion.

  17. A description of chloride cell and kidney tubule alterations in the flatfish Solea senegalensis exposed to moderately contaminated sediments from the Sado estuary (Portugal)

    Science.gov (United States)

    Costa, Pedro M.; Caeiro, Sandra; Diniz, Mário S.; Lobo, Jorge; Martins, Marta; Ferreira, Ana M.; Caetano, Miguel; Vale, Carlos; DelValls, T. Ángel; Costa, M. Helena

    2010-11-01

    The effects of sediment-bound contaminants on kidney and gill chloride cells were surveyed in juvenile Solea senegalensis exposed to fresh sediments collected from three distinct sites of the Sado Estuary (Portugal) in a 28-day laboratorial assay. Sediments were analyzed for metallic contaminants, polycyclic aromatic hydrocarbons and organochlorines as well as for total organic matter, redox potential and fine fraction. The potential for causing adverse biological effects of each surveyed sediment was assessed by comparison of contaminant levels to available guidelines for coastal sediments, namely the Threshold Effects Level ( TEL) and the Probable Effects Level ( PEL). The Sediment Quality Guideline Quotient indices ( SQGQ) were calculated to compare the overall contamination levels of the three stations. A qualitative approach was employed to analyze the histo/cytopathological traits in gill chloride cells and body kidney of fish exposed to each tested sediment for 0, 14 and 28 days. The results showed that sediment contamination can be considered low to moderate and that the least contaminated sediment (from a reference site, with the lowest SQGQ) caused lesser changes in the surveyed organs. However, the most contaminated sediment (by both metallic and organic xenobiotics, with highest SQGQ) was neither responsible for the highest mortality nor for the most pronounced lesions. Exposure to the sediment presenting an intermediate SQGQ, essentially contaminated by organic compounds, caused the highest mortality (48%) and the most severe damage to kidneys, up to full renal necrosis. Chloride cell alterations were similar in fish exposed to the two most contaminated sediments and consisted of a pronounced cellular hypertrophy, likely involving fluid retention and loss of mitochondria. It can be concluded that sediment contamination considered to be low or moderate may be responsible for severe injury to cells and parenchyma involved in the maintenance of osmotic

  18. Sodium pumps in the Malpighian tubule of Rhodnius sp.

    Directory of Open Access Journals (Sweden)

    CARUSO-NEVES CELSO

    2000-01-01

    Full Text Available Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+ATPase activity. Taken together these data indicate that (Na+ + K+ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.

  19. Malpighian tubule development in the red flour beetle (Tribolium castaneum).

    Science.gov (United States)

    King, Benedict; Denholm, Barry

    2014-11-01

    Malpighian tubules (MpTs) are the major organ for excretion and osmoregulation in most insects. MpT development is characterised for Drosophila melanogaster, but not other species. We therefore do not know the extent to which the MpT developmental programme is conserved across insects. To redress this we provide a comprehensive description of MpT development in the beetle Tribolium castaneum (Coleoptera), a species separated from Drosophila by >315 million years. We identify similarities with Drosophila MpT development including: 1) the onset of morphological development, beginning when tubules bud from the gut and proliferate to increase organ size. 2) the tubule is shaped by convergent-extension movements and oriented cell divisions. 3) differentiated tip cells activate EGF-signalling in distal MpT cells through the ligand Spitz. 4) MpTs contain two main cell types - principal and stellate cells, differing in morphology and gene expression. We also describe development of the beetle cryptonephridial system, an adaptation for water conservation, which represents a major modification of the MpT ground plan characterised by intimate association between MpTs and rectum. This work establishes a new model to compare MpT development across insects, and provides a framework to help understand how an evolutionary novelty - the cryptonephridial system - arose during organ evolution.

  20. In vivo model for microbial invasion of tooth root dentinal tubules.

    Science.gov (United States)

    Brittan, Jane L; Sprague, Susan V; Macdonald, Emma L; Love, Robert M; Jenkinson, Howard F; West, Nicola X

    2016-04-01

    Objective Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Material and Methods Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF). DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database. Results All root dentine samples with patent tubules showed evidence of bacterial cell invasion (TIF value range from 5.7 to 9.0) to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. Conclusion A novel in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine.

  1. The postnatal maturation of efferent tubules in the rat: a light and electron microscopy study.

    Science.gov (United States)

    Francavilla, S; Moscardelli, S; Bruno, B; Barcellona, P S; De Martino, C

    1986-07-01

    The postnatal maturation of the epithelium and tubule wall of efferent tubules in the rat was investigated by light and transmission electron microscopy, from birth to 50 days of age, when sperms were released from the seminiferous tubules and appeared in the genital duct. At the end of the first week of life, an endocytotic apparatus is differentiated in the epithelial cells. During the third week of life, efferent tubules developed specializations for the transport of sperms and fluids, namely the appearance of ciliated elements interspersed among the principal cells of the epithelium, and differentiation of myoid elements in the tubule wall. The appearance of specializations related to endocytosis and fluid transport across the epithelium preceded the canalization of the seminiferous cords which, in fact, is reported to appear at the end of the second week of life in the rat, along with the initial secretion of testicular fluid. This suggested that the maturation of efferent tubules is not triggered by the passage of testicular fluid, as surmised for the postnatal differentiation of caput epididymis. The postnatal maturation of efferent tubules was almost complete 35 days after birth. The appearance of sperms in the genital duct of 50-day-old animals was not associated with any remarkable structural change.

  2. In vivo model for microbial invasion of tooth root dentinal tubules

    Directory of Open Access Journals (Sweden)

    Jane L. BRITTAN

    2016-04-01

    Full Text Available ABSTRACT Objective Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Material and Methods Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF. DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database. Results All root dentine samples with patent tubules showed evidence of bacterial cell invasion (TIF value range from 5.7 to 9.0 to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. Conclusion A novel in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine.

  3. Analysis of chromatin compaction and determination of the storage time and distribution of a rooster’s (Gallus gallus spermatozoa in the sperm storage tubules of hens

    Directory of Open Access Journals (Sweden)

    Ana Carolina Nunes Rodrigues

    2011-06-01

    Full Text Available The objectives of this work were to elucidate alterations in the compaction of sperm chromatin during storage in the sperm storage tubules of fowl hens, to observe the storage time, and to estimate the variation in the amount of stored spermatozoa in the cranial, medium and caudal sections of the uterovaginal junction over a period of 23 days. For the analysis of the spermatozoa during storage in the sperm storage tubules of hens, 48 thirty-six-week-old meat chickens of the Cobb Avian 48 strain were used. Samples from the uterovaginal junction of the hens were collected for 23 days after the mating with the roosters, and six hens were euthanized every four days for the production of histological microscope slides. Spermatozoa were found throughout the area of the sperm storage tubules. Most of them were gathered in the medium section of the uterovaginal junction. There was no meaningful statistical difference in the amount of sperm in the sperm storage tubules over the days of the experiment. A greater amount of sperm cells were observed up to the 23rd day of assessment. Therefore, the storage of spermatozoa in hens lasts at least 23 days.

  4. Collective cell migration: Implications for wound healing and cancer invasion

    Directory of Open Access Journals (Sweden)

    Li Li

    2013-07-01

    Full Text Available During embryonic morphogenesis, wound repair and cancer invasion, cells often migrate collectively via tight cell-cell junctions, a process named collective migration. During such migration, cells move as coherent groups, large cell sheets, strands or tubes rather than individually. One unexpected finding regarding collective cell migration is that being a "multicellular structure" enables cells to better respond to chemical and physical cues, when compared with isolated cells. This is important because epithelial cells heal wounds via the migration of large sheets of cells with tight intercellular connections. Recent studies have gained some mechanistic insights that will benefit the clinical understanding of wound healing in general. In this review, we will briefly introduce the role of collective cell migration in wound healing, regeneration and cancer invasion and discuss its underlying mechanisms as well as implications for wound healing.

  5. Rho GTPases in collective cell migration

    NARCIS (Netherlands)

    Zegers, M.M.; Friedl, P.

    2014-01-01

    The family of Rho GTPases are intracellular signal transducers that link cell surface signals to multiple intracellular responses. They are best known for their role in regulating actin dynamics required for cell migration, but in addition control cell-cell adhesion, polarization, vesicle traffickin

  6. Rho GTPases in collective cell migration

    NARCIS (Netherlands)

    Zegers, M.M.; Friedl, P.

    2014-01-01

    The family of Rho GTPases are intracellular signal transducers that link cell surface signals to multiple intracellular responses. They are best known for their role in regulating actin dynamics required for cell migration, but in addition control cell-cell adhesion, polarization, vesicle

  7. Development of Pulsating Tubules with Chiral Inversion

    Science.gov (United States)

    2013-09-21

    assemblies, such as tubules, toroids , porous capsules, and helical fibers, by adjusting the relative volume fraction between hydrophobic and hydrophilic...1 could change their shape into helical tubules at higher and discrete macrocycles at lower concentrations. Metal- containing macrocycles were found to...this direction of research in mind, we synthesized self-assembling molecules 1 and 2 consisting of a long bent-shaped aromatic segment containing m

  8. Biodegradable chitin conduit tubulation combined with bone marrow mesenchymal stem cell transplantation for treatment of spinal cord injury by reducing glial scar and cavity formation

    Institute of Scientific and Technical Information of China (English)

    Feng Xue; Er-jun Wu; Pei-xun Zhang; Li-ya A; Yu-hui Kou; Xiao-feng Yin; Na Han

    2015-01-01

    We examined the restorative effect of modiifed biodegradable chitin conduits in combination with bone marrow mesenchymal stem cell transplantation after right spinal cord hemisection injury. Immunohistochemical staining revealed that biological conduit sleeve bridging reduced glial scar formation and spinal muscular atrophy after spinal cord hemisection. Bone marrow mesenchymal stem cells survived and proliferated after transplantationin vivo, and differentiated into cells double-positive for S100 (Schwann cell marker) and glial ifbrillary acidic protein (glial cell marker) at 8 weeks. Retrograde tracing showed that more nerve ifbers had grown through the injured spinal cord at 14 weeks after combination therapy than either treatment alone. Our ifndings indicate that a biological conduit combined with bone marrow mesenchymal stem cell transplantation effectively prevented scar formation and provided a favorable local microenvi-ronment for the proliferation, migration and differentiation of bone marrow mesenchymal stem cells in the spinal cord, thus promoting restoration following spinal cord hemisection injury.

  9. The development of enamel tubules during the formation of enamel in the marsupial Monodelphis domestica.

    OpenAIRE

    Sasagawa, I; Ferguson, M W

    1991-01-01

    In Monodelphis domestica, although both processes from odontoblasts and projections from ameloblasts were found in developing enamel, the majority of the contents of enamel tubules were probably processes that originated from odontoblasts. Processes from odontoblasts penetrating into enamel touched part of the ameloblasts in the stage of enamel formation. No specialised cell junctions were seen at the adherence between the two. There were no enamel tubules in the aprismatic and pseudoprismati...

  10. Cubilin Is Essential for Albumin Reabsorption in the Renal Proximal Tubule

    OpenAIRE

    Amsellem, S.; Gburek, J.; Hamard, G.; Nielsen, R.; Willnow, T.E.; Devuyst, O.; Nexo, E.; Verroust, P. J.; Christensen, E I; Kozyraki, R.

    2010-01-01

    Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells d...

  11. Calcium Oxalate Accumulation in Malpighian Tubules of Silkworm (Bombyx mori)

    Science.gov (United States)

    Wyman, Aaron J.; Webb, Mary Alice

    2007-04-01

    Silkworm provides an ideal model system for study of calcium oxalate crystallization in kidney-like organs, called Malpighian tubules. During their growth and development, silkworm larvae accumulate massive amounts of calcium oxalate crystals in their Malpighian tubules with no apparent harm to the organism. This manuscript reports studies of crystal structure in the tubules along with analyses identifying molecular constituents of tubule exudate.

  12. Emergent collective chemotaxis without single-cell gradient sensing

    CERN Document Server

    Camley, Brian A; Levine, Herbert; Rappel, Wouter-Jan

    2015-01-01

    Many eukaryotic cells chemotax, sensing and following chemical gradients. However, even if single cells do not chemotax significantly, small clusters may still follow a gradient; this behavior is observed in neural crest cells and during border cell migration in Drosophila, but its origin remains puzzling. Here, we study this "collective guidance" analytically and computationally. We show collective chemotaxis can exist without single-cell chemotaxis if contact inhibition of locomotion (CIL), where cells polarize away from cell-cell contact, is regulated by the chemoattractant. We present explicit formulas for how cluster velocity and chemotactic index depend on the number and organization of cells in the cluster. Pairs of cells will have velocities that are strongly dependent on the cell pair's orientation: this provides a simple test for the presence of collective guidance in neural crest cells and other systems. We also study cluster-level adaptation, amplification, and cohesion via co-attraction.

  13. 75 FR 11545 - Agency Information Collection Activities; Proposed Collection; Comment Request; Human Cells...

    Science.gov (United States)

    2010-03-11

    ... blood stem cell, stem cell products from cord blood, reproductive tissue, and sperm banks), including... Collection; Comment Request; Human Cells, Tissues, and Cellular and Tissue-Based Products: Establishment... regulations related to human cells, tissues, and cellular and tissue-based products (HCT Ps)...

  14. Collective cell movement promotes synchronization of coupled genetic oscillators.

    Science.gov (United States)

    Uriu, Koichiro; Morelli, Luis G

    2014-07-15

    Collective cell movement is a crucial component of embryonic development. Intercellular interactions regulate collective cell movement by allowing cells to transfer information. A key question is how collective cell movement itself influences information flow produced in tissues by intercellular interactions. Here, we study the effect of collective cell movement on the synchronization of locally coupled genetic oscillators. This study is motivated by the segmentation clock in zebrafish somitogenesis, where short-range correlated movement of cells has been observed. We describe the segmentation clock tissue by a Voronoi diagram, cell movement by the force balance of self-propelled and repulsive forces between cells, the dynamics of the direction of self-propelled motion, and the synchronization of genetic oscillators by locally coupled phase oscillators. We find that movement with a correlation length of about 2 ∼ 3 cell diameters is optimal for the synchronization of coupled oscillators. Quantification of cell mixing reveals that this short-range correlation of cell movement allows cells to exchange neighbors most efficiently. Moreover, short-range correlated movement strongly destabilizes nonuniform spatial phase patterns, further promoting global synchronization. Our theoretical results suggest that collective cell movement may enhance the synchronization of the segmentation clock in zebrafish somitogenesis. More generally, collective cell movement may promote information flow in tissues by enhancing cell mixing and destabilizing spurious patterns.

  15. Staphylococcus aureus Alpha-Toxin Induces the Formation of Dynamic Tubules Labeled with LC3 within Host Cells in a Rab7 and Rab1b-Dependent Manner

    Directory of Open Access Journals (Sweden)

    María M. López de Armentia

    2017-10-01

    Full Text Available Staphylococcus aureus is a pathogen that causes severe infectious diseases that eventually lead to septic and toxic shock. S. aureus infection is characterized by the production of virulence factors, including enzymes and toxins. After internalization S. aureus resides in a phagosome labeled with Rab7 protein. Here, we show that S. aureus generates tubular structures marked with the small GTPases Rab1b and Rab7 and by the autophagic protein LC3 at early times post-infection. As shown by live cell imaging these tubular structures are highly dynamic, extend, branch and grow in length. We have named them S. aureus induced filaments (Saf. Furthermore, we demonstrate that the formation of these filaments depends on the integrity of microtubules and the activity of the motor protein Kinesin-1 (Kif5B and the Rab-interacting lysosomal protein (RILP. Our group has previously reported that α-hemolysin, a secreted toxin of S. aureus, is responsible of the activation of the autophagic pathway induced by the bacteria. In the present report, we demonstrate that the autophagic protein LC3 is recruited to the membrane of S. aureus induced filaments and that α-hemolysin is the toxin that induces Saf formation. Interestingly, increasing the levels of intracellular cAMP significantly inhibited Saf biogenesis. Remarkably in this report we show the formation of tubular structures that emerge from the S. aureus-containing phagosome and that these tubules generation seems to be required for efficient bacteria replication.

  16. Hypertension-linked mutation of α-adducin increases CFTR surface expression and activity in HEK and cultured rat distal convoluted tubule cells.

    Science.gov (United States)

    Mondini, Anna; Sassone, Francesca; Civello, Davide Antonio; Garavaglia, Maria Lisa; Bazzini, Claudia; Rodighiero, Simona; Vezzoli, Valeria; Conti, Fabio; Torielli, Lucia; Capasso, Giovanbattista; Paulmichl, Markus; Meyer, Giuliano

    2012-01-01

    The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

  17. Hypertension-linked mutation of α-adducin increases CFTR surface expression and activity in HEK and cultured rat distal convoluted tubule cells.

    Directory of Open Access Journals (Sweden)

    Anna Mondini

    Full Text Available The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats, an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus hypertensive model carrying the F316Y adducin mutation, compared to MNS (Milan Normotensive Strain rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

  18. Collective Movement of Epithelial Cells on a Collagen Gel Substrate

    OpenAIRE

    Haga, Hisashi; Irahara, Chikako; KOBAYASHI, Ryo; Nakagaki, Toshiyuki; Kawabata, Kazushige

    2004-01-01

    Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, th...

  19. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Lee, Sang Yong [Department of Diagnostic Radiology, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Han, Myung Kwan [Department of Microbiology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kim, Duk Hoon [Division of Forensic Medicine, National Forensic Service, Seoul (Korea, Republic of); Kim, Won, E-mail: kwon@jbnu.ac.kr [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  20. Occluding effect of dentifrices on dentinal tubules.

    Science.gov (United States)

    Arrais, César Augusto Galvão; Micheloni, Carolina Diniz; Giannini, Marcelo; Chan, Daniel C N

    2003-11-01

    The objective of this study was to evaluate the tubule occluding ability of three commercial available dentifrices (Sensodyne, Emoform and Sorriso) by Scanning Electron Microscopy. Fifty cervical areas from buccal and lingual surfaces of sound third human molars were used. Cervical enamel was wet abraded to expose flat dentin surfaces and further polished with diamond pastes. Specimens were randomly divided into five groups (n=10): G1-no brushing; G2-brushing without dentifrice; G3-brushing with Sensodyne; G4-brushing with Emoform; G5-brushing with Sorriso. Brushed specimens were treated for 4 min per day, for 7 days in a toothbrushing machine. Specimens were prepared and observed under SEM for calculation of the percentage of occluded tubules. In addition, slurries of toothpastes were analyzed by X-ray microanalysis. Data were statistically analyzed by ANOVA and Tukey test (p<0.05). Means of occluded tubules in descending order were: G5-98.83+/-3.31% (a), G4-96.02+/-5.24% (a), G3-80.12+/-24.65% (a), G1-37.31+/-24.22% (b); G2-22.92+/-15.19% (b). The three tested dentifrices produced increased dentinal occlusion as compared to controls (p<0.05) but equivalent occlusion among each other. Calcium from calcium-carbonate abrasive was identified in all dentifrices. Results indicated that the use of all dentifrices occluded tubules more than no brushing and brushing without dentifrices groups. Thus, the tested dentifrices seem effective for desensitization by tubule occlusion.

  1. The stress response of human proximal tubule cells to cadmium involves up-regulation of haemoxygenase 1 and metallothionein but not cytochrome P450 enzymes.

    Science.gov (United States)

    Boonprasert, Kanyarat; Satarug, Soisungwan; Morais, Christudas; Gobe, Glenda C; Johnson, David W; Na-Bangchang, Kesara; Vesey, David A

    2016-05-13

    Enzymes of the cytochrome P450 (CYP) super-family are implicated in cadmium (Cd) -induced nephrotoxicity, however, direct evidence is lacking. This study investigated the endogenous expression of various CYP proteins together with the stress-response proteins, heme oxygenase-1 (HO-1) and metallothionein (MT) in human kidney sections and in cadmium-exposed primary cultures of human proximal tubular epithelial cells (PTC). By immunohistochemistry, the CYP members 2B6, 4A11 and 4F2 were prominently expressed in the cortical proximal tubular cells and to a lesser extent in distal tubular cells. Low levels of CYPs 2E1 and 3A4 were also detected. In PTC, in the absence of Cd, CYP2E1, CYP3A4, CYP4F2 and MT were expressed, but HO-1, CYP2B6 and CYP4A11 were not detected. A range of cadmium concentrations (0-100μM) were utilized to induce stress conditions. MT protein was further induced by as little as 0.5μM cadmium, reaching a 6-fold induction at 20μM, whereas for HO-1, a 5μM cadmium concentration was required for initial induction and at 20μM cadmium reached a 15-fold induction. The expression of CYP2E1, CYP3A4, and CYP4F2 were not altered by any cadmium concentrations tested at 48h. Cadmium caused a reduction in cell viability at concentrations above 10μM. In conclusion although cultured PTC, do express CYP proteins, (CYP2E1, CYP3A4, and CYP4F2), Cd-induced cell stress as indicted by induction of HO-1 and MT does not alter expression of these CYP proteins at 48h. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Silk film topography directs collective epithelial cell migration.

    Directory of Open Access Journals (Sweden)

    Brian D Lawrence

    Full Text Available The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography's edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.

  3. Maturational changes in connexin 43 expression in the seminiferous tubules may depend on thyroid hormone action

    Science.gov (United States)

    Marchlewska, Katarzyna; Kula, Krzysztof; Walczak-Jedrzejowska, Renata; Kula, Wojciech; Oszukowska, Elzbieta; Filipiak, Eliza; Moszura, Tomasz

    2013-01-01

    Introduction Connexin 43 (Cx43) mediates the effect of thyroid hormone on Sertoli cell maturation in vitro. We investigated the influence of triiodothyronine (T3) administration on Cx43 expression in relation to the progress in seminiferous tubule maturation. Material and methods Male rats were daily injected with 100 µg T3/kg body weight from birth until postnatal day (pnd) 5 (transient treatment – tT3) or until pnd 15 (continuous treatment – cT3) or solvent – control (C). On pnd 16 serum hormone levels, body and testes weight, seminiferous tubule morphometry, Cx43 immunostaining and germ cell degeneration were investigated. Cx43 expression was also assessed in six 50-day-old adult untreated rats. Result tT3 increased 2.6-fold serum level of T3, testes weight, and seminiferous tubule diameter, and induced maturation-like dislocation of Cx43 expression from the apical to the peripheral region of Sertoli cell cytoplasm. In addition, incidence of Cx43-positive tubules declined from 86% in C to 46% after tT3, being similar to the adult value (30% of tubules Cx43-positive). In turn, cT3 increased serum T3 level 12-fold, and decreased body weight. Seminiferous tubules became shortened and distended, Sertoli cell cytoplasm vacuolated, Cx43 expression had minimal intensity and germ cell degeneration increased. Conclusions Cx43 might intermediate a short and transient stimulatory effect of T3 on seminiferous tubule maturation that disappeared together with exposure to the toxic effect of a continuously high level of the hormone. PMID:23515877

  4. Loss of NHERF-1 expression prevents dopamine-mediated Na-K-ATPase regulation in renal proximal tubule cells from rat models of hypertension: aged F344 rats and spontaneously hypertensive rats.

    Science.gov (United States)

    Barati, Michelle T; Ketchem, Corey J; Merchant, Michael L; Kusiak, Walter B; Jose, Pedro A; Weinman, Edward J; LeBlanc, Amanda J; Lederer, Eleanor D; Khundmiri, Syed J

    2017-08-01

    Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K(+) transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

  5. Troglitazone induced cytosolic acidification via extracellular signal-response kinase activation and mitochondrial depolarization: complex I proton pumping regulates ammoniagenesis in proximal tubule-like LLC-PK1 cells.

    Science.gov (United States)

    Oliver, Robert; Friday, Ellen; Turturro, Francesco; Welbourne, Tomas

    2008-01-01

    proton pumping into the cytosol while chronically Complex I activity appears coupled to mitochondrial glutamate uptake and oxidation to ammonium at the expense of cytosolic transamination and alanine formation in these proximal tubule-like cells. Copyright 2008 S. Karger AG, Basel.

  6. Telmisartan counteracts TGF-β1 induced epithelial–to–mesenchymal transition via PPAR-γ in human proximal tubule epithelial cells

    Science.gov (United States)

    Chen, Yumin; Luo, Qiong; Xiong, Zibo; Liang, Wei; Chen, Li; Xiong, Zuying

    2012-01-01

    Chronic renal failure (CRF) mainly results from kidney fibrosis. Epithelial-to-mesenchymal transition (EMT) occurs in stressed tubular epithelial cells and contributes to renal fibrosis. Transforming growth factor-β1 (TGF-β1) has been shown to initiate and complete the whole EMT process. Peroxisome proliferators-activated receptor-γ (PPAR-γ) exerts anti-inflammatory, anti-fibrotic and vaculo-protective effects on different renal diseases. Telmisartan is a member of angiotensin II (Ang II) receptor blocker (ARB) family. Recent studies show that Telmisartan has a partial agonistic effect on PPAR-γ. Therefore, we tested the hypothesis that Telmisartan reverses the progression of induced EMT by TGF-β1 in cultured human renal proximal tubular epithelial (HK-2) cells. Cultured HK-2 cells were treated with TGF-β1 (3 ng/ml), a combination of TGF-β1 and Telmisartan (10-200umol/L) and a combination of TGF-β1, Telmisartan and GW9662, a PPAR-γ antagonist for 48 hours. EMT was determined by quantitative real-time PCR analysis of E-cadherin (E-cad), Connective Tissue Growth Factor (CTGF) and PPAR-γ transcript expression and immunocytochemical analysis of E-cad, α-Smooth Muscle Actin (α-SMA) and PPAR-γ protein expression. TGF-β1 induced phenotypic EMT in cultured HK-2 cell line via significantly reduced E-cad expression and significantly increased CTGF, α-SMA expression in association with the loss of epithelial morphology. Telmisartan reversed all EMT markers in a dose-dependent manner which was inhibited by PPAR antagonist GW9662. In the present study, it was suggested that Telmisartan attenuated TGF-β1 induced EMT by agonistic activation of PPAR-γ. PMID:22949934

  7. Invasion of dentinal tubules by oral bacteria.

    Science.gov (United States)

    Love, R M; Jenkinson, H F

    2002-01-01

    Bacterial invasion of dentinal tubules commonly occurs when dentin is exposed following a breach in the integrity of the overlying enamel or cementum. Bacterial products diffuse through the dentinal tubule toward the pulp and evoke inflammatory changes in the pulpo-dentin complex. These may eliminate the bacterial insult and block the route of infection. Unchecked, invasion results in pulpitis and pulp necrosis, infection of the root canal system, and periapical disease. While several hundred bacterial species are known to inhabit the oral cavity, a relatively small and select group of bacteria is involved in the invasion of dentinal tubules and subsequent infection of the root canal space. Gram-positive organisms dominate the tubule microflora in both carious and non-carious dentin. The relatively high numbers of obligate anaerobes present-such as Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., Peptostreptococcus micros, and Veillonella spp.-suggest that the environment favors growth of these bacteria. Gram-negative obligate anaerobic rods, e.g., Porphyromonas spp., are less frequently recovered. Streptococci are among the most commonly identified bacteria that invade dentin. Recent evidence suggests that streptococci may recognize components present within dentinal tubules, such as collagen type I, which stimulate bacterial adhesion and intra-tubular growth. Specific interactions of other oral bacteria with invading streptococci may then facilitate the invasion of dentin by select bacterial groupings. An understanding the mechanisms involved in dentinal tubule invasion by bacteria should allow for the development of new control strategies, such as inhibitory compounds incorporated into oral health care products or dental materials, which would assist in the practice of endodontics.

  8. Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle.

    Science.gov (United States)

    Demonbreun, Alexis R; Swanson, Kaitlin E; Rossi, Ann E; Deveaux, H Kieran; Earley, Judy U; Allen, Madison V; Arya, Priyanka; Bhattacharyya, Sohinee; Band, Hamid; Pytel, Peter; McNally, Elizabeth M

    2015-01-01

    We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.

  9. Eps 15 Homology Domain (EHD-1 Remodels Transverse Tubules in Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Alexis R Demonbreun

    Full Text Available We previously showed that Eps15 homology domain-containing 1 (EHD1 interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.

  10. Membrane tubule formation by banana-shaped proteins with or without transient network structure

    Science.gov (United States)

    Noguchi, Hiroshi

    2016-02-01

    In living cells, membrane morphology is regulated by various proteins. Many membrane reshaping proteins contain a Bin/Amphiphysin/Rvs (BAR) domain, which consists of a banana-shaped rod. The BAR domain bends the biomembrane along the rod axis and the features of this anisotropic bending have recently been studied. Here, we report on the role of the BAR protein rods in inducing membrane tubulation, using large-scale coarse-grained simulations. We reveal that a small spontaneous side curvature perpendicular to the rod can drastically alter the tubulation dynamics at high protein density, whereas no significant difference is obtained at low density. A percolated network is intermediately formed depending on the side curvature. This network suppresses tubule protrusion, leading to the slow formation of fewer tubules. Thus, the side curvature, which is generated by protein-protein and membrane-protein interactions, plays a significant role in tubulation dynamics. We also find that positive surface tensions and the vesicle membrane curvature can stabilize this network structure by suppressing the tubulation.

  11. Emergent collective chemotaxis without single-cell gradient sensing

    Science.gov (United States)

    Camley, Brian A.; Zimmermann, Juliane; Levine, Herbert; Rappel, Wouter-Jan

    2016-01-01

    Many eukaryotic cells chemotax, sensing and following chemical gradients. However, experiments have shown that even under conditions when single cells cannot chemotax, small clusters may still follow a gradient. This behavior has been observed in neural crest cells, in lymphocytes, and during border cell migration in Drosophila, but its origin remains puzzling. Here, we propose a new mechanism underlying this “collective guidance”, and study a model based on this mechanism both analytically and computationally. Our approach posits that contact inhibition of locomotion (CIL), where cells polarize away from cell-cell contact, is regulated by the chemoattractant. Individual cells must measure the mean attractant value, but need not measure its gradient, to give rise to directional motility for a cell cluster. We present analytic formulas for how cluster velocity and chemotactic index depend on the number and organization of cells in the cluster. The presence of strong orientation effects provides a simple test for our theory of collective guidance. PMID:26991203

  12. Asymmetric division coordinates collective cell migration in angiogenesis.

    Science.gov (United States)

    Costa, Guilherme; Harrington, Kyle I; Lovegrove, Holly E; Page, Donna J; Chakravartula, Shilpa; Bentley, Katie; Herbert, Shane P

    2016-12-01

    The asymmetric division of stem or progenitor cells generates daughters with distinct fates and regulates cell diversity during tissue morphogenesis. However, roles for asymmetric division in other more dynamic morphogenetic processes, such as cell migration, have not previously been described. Here we combine zebrafish in vivo experimental and computational approaches to reveal that heterogeneity introduced by asymmetric division generates multicellular polarity that drives coordinated collective cell migration in angiogenesis. We find that asymmetric positioning of the mitotic spindle during endothelial tip cell division generates daughters of distinct size with discrete 'tip' or 'stalk' thresholds of pro-migratory Vegfr signalling. Consequently, post-mitotic Vegfr asymmetry drives Dll4/Notch-independent self-organization of daughters into leading tip or trailing stalk cells, and disruption of asymmetry randomizes daughter tip/stalk selection. Thus, asymmetric division seamlessly integrates cell proliferation with collective migration, and, as such, may facilitate growth of other collectively migrating tissues during development, regeneration and cancer invasion.

  13. Dentin tubule occluding ability of dentin desensitizers.

    Science.gov (United States)

    Han, Linlin; Okiji, Takashi

    2015-04-01

    To compare the dentin tubule-occluding ability of fluoroaluminocalciumsilicate-based (Nanoseal), calcium phosphate-based (Teethmate Desensitizer), resin-containing oxalate (MS Coat ONE) and diamine silver fluoride (Saforide) dentin desensitizers using artificially demineralized bovine dentin. Simulated hypersensitive dentin was created using cervical dentin sections derived from bovine incisors using phosphoric acid etching followed by polishing with a paste containing hydroxyapatite. The test desensitizers were applied in one, two, or three cycles, where each cycle involved desensitizer application, brushing, and immersion in artificial saliva (n= 5 each). The dentin surfaces were examined with scanning electron microscopy, and the dentin tubule occlusion rate was calculated. The elemental composition of the deposits was analyzed with electron probe microanalysis. Data were analyzed by one-way ANOVA and the Tukey honestly significant different test. Marked deposit formation was observed on the specimens treated with Nanoseal or Teethmate Desensitizer, and tags were detected in the specimens' dentin tubules. These findings became more prominent as the number of application cycles increased. The major elemental components of the tags were Ca, F, and Al (Nanoseal) and Ca and P (Teethmate Desensitizer). The tubule occlusion rates of MS Coat ONE and Saforide were significantly lower than those of Nanoseal and Teethmate Desensitizer (P< 0.05).

  14. Collisions of deformable cells lead to collective migration

    Science.gov (United States)

    Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

    2015-03-01

    Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility - acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.

  15. Ecdysone regulates morphogenesis and function of Malpighian tubules in Drosophila melanogaster through EcR-B2 isoform.

    Science.gov (United States)

    Gautam, Naveen Kumar; Verma, Puja; Tapadia, Madhu G

    2015-02-15

    Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. We have conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and β-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na(+)/K(+)-ATPase pump. B2 isoform of ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development.

  16. Modelling Rho GTPase biochemistry to predict collective cell migration

    Science.gov (United States)

    Merchant, Brian; Feng, James

    The collective migration of cells, due to individual cell polarization and intercellular contact inhibition of locomotion, features prominently in embryogenesis and metastatic cancers. Existing methods for modelling collectively migrating cells tend to rely either on highly abstracted agent-based models, or on continuum approximations of the group. Both of these frameworks represent intercellular interactions such as contact inhibition of locomotion as hard-coded rules defining model cells. In contrast, we present a vertex-dynamics framework which predicts polarization and contact inhibition of locomotion naturally from an underlying model of Rho GTPase biochemistry and cortical mechanics. We simulate the interaction between many such model cells, and study how modulating Rho GTPases affects migratory characteristics of the group, in the context of long-distance collective migration of neural crest cells during embryogenesis.

  17. Fluid transport and ion fluxes in mammalian kidney proximal tubule: a model analysis of isotonic transport

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Møbjerg, N.; Sørensen, J. N.

    2006-01-01

    Aim: By mathematical modelling, we analyse conditions for near-isotonic and isotonic transport by mammalian kidney proximal tubule. Methods: The model comprises compliant lateral intercellular space (lis) and cells, and infinitely large luminal and peritubular compartments with diffusible species......: Na+, K+, Cl- and an intracellular non-diffusible anion. Unknown model variables are solute concentrations, electrical potentials, volumes and hydrostatic pressures in cell and lis, and transepithelial potential. We used data mainly from rat proximal tubule to model epithelial cells and interspace...... transport similar to rat proximal tubule. Na+ recirculation is required for truly isotonic transport. The tonicity of the absorbate and the recirculation flux depend critically on ion permeabilities of interspace basement membrane. Conclusion: Our model based on solute-solvent coupling in lateral space...

  18. Fluid transport and ion fluxes in mammalian kidney proximal tubule: a model analysis of isotonic transport

    DEFF Research Database (Denmark)

    Larsen, E.H.; Møbjerg, N.; Sørensen, Jens Nørkær

    2006-01-01

    Aim: By mathematical modelling, we analyse conditions for near-isotonic and isotonic transport by mammalian kidney proximal tubule. Methods: The model comprises compliant lateral intercellular space (lis) and cells, and infinitely large luminal and peritubular compartments with diffusible species......: Na+, K+, Cl and an intracellular non-diffusible anion. Unknown model variables are solute concentrations, electrical potentials, volumes and hydrostatic pressures in cell and lis, and transepithelial potential. We used data mainly from rat proximal tubule to model epithelial cells and interspace...... transport similar to rat proximal tubule. Na+ recirculation is required for truly isotonic transport. The tonicity of the absorbate and the recirculation flux depend critically on ion permeabilities of interspace basement membrane. Conclusion: Our model based on solute-solvent coupling in lateral space...

  19. [N-acetyl-beta-hexosaminidase--marker of damage to renal proximal tubules].

    Science.gov (United States)

    Kepka, Alina; Szajda, Sławomir D; Jankowska, Anna; Waszkiewicz, Napoleon; Chojnowska, Sylwia; Zwierz, Krzysztof

    2008-09-01

    Cells of the renal epithelium synthesize and excrete to urine many enzymes. Among more than 50 enzymes produced by epithelial cells of proximal tubules, only few have a diagnostic value. Determination of the enzymatic activities in urine is sensitive and not invasive method for evaluation the function of renal tubules. Urinary N-acetyl-beta-hexosaminidase (HEX) activity is approved and practically utilized marker of the renal function. HEX is a lysosomal exoglycosidase taking part in catabolism of the sugar chains of glycoconjugates (glycoproteins, glycolipids and proteoglycans). HEX catalyses release of N-acetylglucosamine and N-acetylgalactosamine from a non reducing ends of glycoconjugates. In urine of healthy persons activity of HEX is negligible, but significantly increases after damage to the proximal tubules. The cells of renal proximal tubules are very sensitive to hypoxia. Therefore all renal processes with hypoxia lead to dysfunction of proximal renal tubules and release HEX to urine. Increased activity of HEX in urine was found after intoxication by heavy metals, nephrotoxic drugs, contrast media, fewer, bacterial as well as immunological nephritis and hypertension, diabetes, neoplasms and during renal graft rejection. In the paper we presented review of literature concerning HEX, and its presence in renal tissue and urine, as well as application in diagnostics.

  20. Cell collectivity regulation within migrating cell cluster during Kupffer’s vesicle formation in zebrafish

    Directory of Open Access Journals (Sweden)

    Takaaki eMatsui

    2015-05-01

    Full Text Available Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called collective cell migration, is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as cell collectivity, remain largely unknown. During the formation of Kupffer’s vesicle (KV, an organ of laterality in zebrafish, KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent findings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration.

  1. Antioxidant Protection against Pathological Mycotoxins Alterations on Proximal Tubules in Rat Kidney

    Directory of Open Access Journals (Sweden)

    Susan Abdu

    2011-04-01

    Full Text Available Background: Ochratoxin A (OTA was one of the mycotoxins and received attention worldwide because of the hazard it posed to human and animal health, where the kidney was the primary target organ for OTA toxicity. In the other hand, dates served as a good source of natural antioxidants and could potentially be considered as a functional food.Methods: The study was performed in the department of biology in King Abdulaziz University. Animals were gavage administrated and divided into four groups: first group received (sodium bicarbonate, second group received (289 µg OTA /kg B.W. /day, third group received (1mg Ajwa/kg B.W. / day and fourth group received (289 µg OTA /kg B.W./day+ 1mg Ajwa /kg B.W. / day. Serum (creatinine - urea levels were measured in each group at the time of tissue collection , some biopsies were fixed in 10% buffered formalin solution for light microscopy processing stained with Haematoxylin and Eosin (H& E., Periodic Acid-Schiff (PAS and Masson´s Trichrome (M.T..Other biopsies were immediately collected into electron microscopy processing. Results: After 28 days, a significant decrease in body weight, kidney weight and relative weight was detected in OTA treated group. Also, Serum (creatinine - urea level were elevated .The normal cyto-architecture of proximal tubules were lost exhibiting damaged bruch border, degenerated, binucleated and karyomegalic cells. The most destructed ultra-structure was the mitochondria which severely swollen with disintegrated membranes. In Ajwa Date extract-group the proximal tubules were normal, whereas in Ajwa date extract + OTA -group the severity of the lesions was significantly reduced. Conclusion: The present results indicated that, Ajwa date have protective effects and ameliorated the lesions of Ochratoxin nepherotoxicity which might lead to kidney failure.

  2. Group choreography: mechanisms orchestrating the collective movement of border cells.

    Science.gov (United States)

    Montell, Denise J; Yoon, Wan Hee; Starz-Gaiano, Michelle

    2012-10-01

    Cell movements are essential for animal development and homeostasis but also contribute to disease. Moving cells typically extend protrusions towards a chemoattractant, adhere to the substrate, contract and detach at the rear. It is less clear how cells that migrate in interconnected groups in vivo coordinate their behaviour and navigate through natural environments. The border cells of the Drosophila melanogaster ovary have emerged as an excellent model for the study of collective cell movement, aided by innovative genetic, live imaging, and photomanipulation techniques. Here we provide an overview of the molecular choreography of border cells and its more general implications.

  3. Interdigitated back contact solar cell with high-current collection

    Science.gov (United States)

    Garner, C. M.; Nasby, R. D.; Sexton, F. W.; Rodriguez, J. L.; Norwood, D. P.

    Internal current collection efficiencies greater than 90% and energy conversion efficiencies of 18 % at 30 suns were measured on a laboratory version of the interdigitated back contact (IBC) solar cell. A phosphorous gettering diffusion was performed on the front surface and then etched off to achieve these high current collection efficiencies. Thermal oxides were grown on the front and back of the cell to passivate the silicon surfaces. Although the internal collection efficiencies of the cell were high, series resistance caused the fill factor (FF) to decrease at concentrations above 30 suns. Dark current measurements on cells with a new grid spacing indicate that the series resistance is much lower than in the previous cell design. It is suggested that this should result in higher efficiencies at high concentration.

  4. Functionally induced changes in water transport in the proximal tubule segment of rat kidneys

    DEFF Research Database (Denmark)

    Faarup, Poul; von Holstein-Rathlou, Niels-Henrik; Nørgaard, Tove

    2011-01-01

    To eliminate freezing artifacts in the proximal tubule cells, two cryotechniques were applied to normal rat kidneys, ie, freeze substitution and special freeze drying. In addition, salt depletion and salt loading were applied to groups of rats to evaluate whether the segmental structure of the pr......To eliminate freezing artifacts in the proximal tubule cells, two cryotechniques were applied to normal rat kidneys, ie, freeze substitution and special freeze drying. In addition, salt depletion and salt loading were applied to groups of rats to evaluate whether the segmental structure...

  5. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips

    Science.gov (United States)

    Homan, Kimberly A.; Kolesky, David B.; Skylar-Scott, Mark A.; Herrmann, Jessica; Obuobi, Humphrey; Moisan, Annie; Lewis, Jennifer A.

    2016-10-01

    Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand.

  6. Collection of hematopoietic stem cells from patients with autoimmune diseases

    NARCIS (Netherlands)

    Burt, RK; Fassas, A; Snowden, JA; Kozak, T; Wulffraat, NM; Nash, RA; Dunbar, CE; Arnold, R; Prentice, G; Bingham, S; Marmont, AM; McSweeney, PA; van Laar, J.M.

    We reviewed data from 24 transplant centers in Asia, Australia, Europe, and North America to determine the outcomes of stem cell collection including methods used, cell yields, effects on disease activity, and complications in patients with autoimmune diseases. Twenty-one unprimed bone marrow

  7. Collection of hematopoietic stem cells from patients with autoimmune diseases

    NARCIS (Netherlands)

    Burt, RK; Fassas, A; Snowden, JA; Kozak, T; Wulffraat, NM; Nash, RA; Dunbar, CE; Arnold, R; Prentice, G; Bingham, S; Marmont, AM; McSweeney, PA; van Laar, J.M.

    2001-01-01

    We reviewed data from 24 transplant centers in Asia, Australia, Europe, and North America to determine the outcomes of stem cell collection including methods used, cell yields, effects on disease activity, and complications in patients with autoimmune diseases. Twenty-one unprimed bone marrow harves

  8. Assessment of mitochondrial membrane potential in proximal tubules after hypoxia-reoxygenation.

    Science.gov (United States)

    Feldkamp, Thorsten; Kribben, Andreas; Weinberg, Joel M

    2005-06-01

    Proximal tubules develop a severe energetic deficit during hypoxia-reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential (DeltaPsim) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in DeltaPsim entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of DeltaPsim for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated DeltaPsim, and ATP hydrolysis-supported DeltaPsim. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsim.

  9. Renal compensation to chronic hypoxic hypercapnia: downregulation of pendrin and adaptation of the proximal tubule

    DEFF Research Database (Denmark)

    de Seigneux, Sophie; Malte, Hans; Dimke, Henrik;

    2007-01-01

    The molecular basis for the renal compensation to respiratory acidosis and specifically the role of pendrin in this condition are unclear. Therefore, we studied the adaptation of the proximal tubule and the collecting duct to respiratory acidosis. Male Wistar-Hannover rats were exposed to either ...

  10. Viable bacteria in root dentinal tubules of teeth with apical periodontitis

    NARCIS (Netherlands)

    Peters, LB; Wesselink, P.R.; Buijs, JF; van Winkelhoff, AJ

    2001-01-01

    Two sets of teeth with apical periodontitis were collected at different geographic locations to study the identity of bacteria left in the root dentinal tubules. Root dentin of 20 of these teeth was cultured from three locations between pulp and cementum (A, B, and C). In addition dentin from eight

  11. Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-kappaB signal pathways in renal proximal tubule cells.

    Science.gov (United States)

    Lee, Yu Jin; Han, Ho Jae

    2008-03-01

    It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [(3)H]thymidine incorporation in a dose (> or =3 mg/ml)- and time (> or =3 h)-dependent manner, intracellular Ca(2+) concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca(2+) chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca(2+) chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor-kappaB (NF-kappaB) and inhibitor of NF-kappaB (IkappaB) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca(2+), PKC, EGFR, p44/42 MAPK, or NF-kappaB signal pathways blocked the BSA-induced incorporation of [(3)H]thymidine. Consequently, the inhibition of Ca(2+), PKC, EGFR, p44/42 MAPKs, or NF-kappaB blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21(WAF/Cip1) and p27(Kip1) expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca(2+)/PKC as well as the EGFR-dependent p44/42 MAPK and NF-kappaB signal pathways in PTCs.

  12. Dentine tubule infection and endodontic therapy implications.

    Science.gov (United States)

    Oguntebi, B R

    1994-07-01

    A critical review of the literature suggests that the microenvironment of dentinal tubules appears to favour the selection of relatively few bacterial types irrespective of the aetiology of the infection process; coronal dental caries or pulpar necrosis. These bacteria may constitute an important reservoir from which root canal infection and reinfection may occur following pulp necrosis or during and after endodontic treatment. Previous studies of this microflora have utilized microbiological culture techniques which need to be supplemented by those that allow in situ demonstration as well as identification of the bacteria. Newer treatment strategies that are designed to eliminate this microflora must include agents that can penetrate the dentinal tubules and destroy these microorganisms, since they are located in an area beyond the host defence mechanisms where they cannot be reached by systemically administered antimicrobial agents.

  13. Effects of TNF-alpha on Endothelial Cell Collective Migration

    Science.gov (United States)

    Chen, Desu; Wu, Di; Helim Aranda-Espinoza, Jose; Losert, Wolfgang

    2013-03-01

    Tumor necrosis factor (TNF-alpha) is a small cell-signaling protein usually released by monocytes and macrophages during an inflammatory response. Previous work had shown the effects of TNF-alpha on single cell morphology, migration, and biomechanical properties. However, the effect on collective migrations remains unexplored. In this work, we have created scratches on monolayers of human umbilical endothelial cells (HUVECs) treated with 25ng/mL TNF-alpha on glass substrates. The wound healing like processes were imaged with phase contrast microscopy. Quantitative analysis of the collective migration of cells treated with TNF-alpha indicates that these cells maintain their persistent motion and alignment better than untreated cells. In addition, the collective migration was characterized by measuring the amount of non-affine deformations of the wound healing monolayer. We found a lower mean non-affinity and narrower distribution of non-affinities upon TNF-alpha stimulation. These results suggest that TNF-alpha introduces a higher degree of organized cell collective migration.

  14. Multi-cellular logistics of collective cell migration.

    Directory of Open Access Journals (Sweden)

    Masataka Yamao

    Full Text Available During development, the formation of biological networks (such as organs and neuronal networks is controlled by multicellular transportation phenomena based on cell migration. In multi-cellular systems, cellular locomotion is restricted by physical interactions with other cells in a crowded space, similar to passengers pushing others out of their way on a packed train. The motion of individual cells is intrinsically stochastic and may be viewed as a type of random walk. However, this walk takes place in a noisy environment because the cell interacts with its randomly moving neighbors. Despite this randomness and complexity, development is highly orchestrated and precisely regulated, following genetic (and even epigenetic blueprints. Although individual cell migration has long been studied, the manner in which stochasticity affects multi-cellular transportation within the precisely controlled process of development remains largely unknown. To explore the general principles underlying multicellular migration, we focus on the migration of neural crest cells, which migrate collectively and form streams. We introduce a mechanical model of multi-cellular migration. Simulations based on the model show that the migration mode depends on the relative strengths of the noise from migratory and non-migratory cells. Strong noise from migratory cells and weak noise from surrounding cells causes "collective migration," whereas strong noise from non-migratory cells causes "dispersive migration." Moreover, our theoretical analyses reveal that migratory cells attract each other over long distances, even without direct mechanical contacts. This effective interaction depends on the stochasticity of the migratory and non-migratory cells. On the basis of these findings, we propose that stochastic behavior at the single-cell level works effectively and precisely to achieve collective migration in multi-cellular systems.

  15. Kidney-on-a-chip technology for renal proximal tubule tissue reconstruction.

    Science.gov (United States)

    Nieskens, Tom T G; Wilmer, Martijn J

    2016-11-05

    The renal proximal tubule epithelium is responsible for active secretion of endogenous and exogenous waste products from the body and simultaneous reabsorption of vital compounds from the glomerular filtrate. The complexity of this transport machinery makes investigation of processes such as tubular drug secretion a continuous challenge for researchers. Currently available renal cell culture models often lack sufficient physiological relevance and reliability. Introducing complex biological culture systems in a 3D microfluidic design improves the physiological relevance of in vitro renal proximal tubule epithelium models. Organ-on-a-chip technology provides a promising alternative, as it allows the reconstruction of a renal tubule structure. These microfluidic systems mimic the in vivo microenvironment including multi-compartmentalization and exposure to fluid shear stress. Increasing data supports that fluid shear stress impacts the phenotype and functionality of proximal tubule cultures, for which we provide an extensive background. In this review, we discuss recent developments of kidney-on-a-chip platforms with current and future applications. The improved proximal tubule functionality using 3D microfluidic systems is placed in perspective of investigating cellular signalling that can elucidate mechanistic aberrations involved in drug-induced kidney toxicity. Copyright © 2016. Published by Elsevier B.V.

  16. Automatic quantitative analysis of t-tubule organization in cardiac myocytes using ImageJ.

    Science.gov (United States)

    Pasqualin, Côme; Gannier, François; Malécot, Claire O; Bredeloux, Pierre; Maupoil, Véronique

    2015-02-01

    The transverse tubule system in mammalian striated muscle is highly organized and contributes to optimal and homogeneous contraction. Diverse pathologies such as heart failure and atrial fibrillation include disorganization of t-tubules and contractile dysfunction. Few tools are available for the quantification of the organization of the t-tubule system. We developed a plugin for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. This plugin (TTorg) analyzes raw confocal microscopy images. Analysis options include the whole image, specific regions of the image (cropping), and z-axis analysis of the same image. Batch analysis of a series of images with identical criteria is also one of the options. There is no need to either reorientate any specimen to the horizontal or to do a thresholding of the image to perform analysis. TTorg includes a synthetic "myocyte-like" image generator to test the plugin's efficiency in the user's own experimental conditions. This plugin was validated on synthetic images for different simulated cell characteristics and acquisition parameters. TTorg was able to detect significant differences between the organization of the t-tubule systems in experimental data of mouse ventricular myocytes isolated from wild-type and dystrophin-deficient mice. TTorg is freely distributed, and its source code is available. It provides a reliable, easy-to-use, automatic, and unbiased measurement of t-tubule organization in a wide variety of experimental conditions.

  17. 多巴胺受体和脂筏对高血压患者细胞NADPH氧化酶的作用%Dopamine receptor and raft lipids regulate NADPH oxidase activity in hypertensive renal proximal tubule cells

    Institute of Scientific and Technical Information of China (English)

    鹿敏; 刘晓颖; 韩卫星

    2013-01-01

    目的 探讨还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH氧化酶即Nox)亚单位在高血压患者肾脏近曲小管细胞中的表达及其活性变化,以及多巴胺受体和脂筏在其中的调节作用.方法 细胞分为正常组和高血压组,未经任何药物刺激的两组细胞分别作为正常对照组和高血压对照组,采用葡萄糖浓度梯度超速离心法提取细胞膜的脂筏和非脂筏区蛋白,经Western blot检测Nox亚单位蛋白的表达,光泽精化学发光法动态测定细胞膜Nox的活性.结果 多巴胺受体激动剂fenoldopam明显减少gp91phox在正常对照组[(17±3.3)%]和高血压对照组[(20±3.4)%,P<0.05]细胞膜脂筏区域的表达,降低正常对照组p22phox[(15±2.0)%,P<0.05]、p67phox、rac1在脂筏区的表达,但不能减少高血压对照组p22phox、p67phox、rac1蛋白的表达;胆固醇耗竭剂β-CD减少正常对照组gp91phox、p22phox在脂筏区的表达,不能减少高血压对照组Nox亚单位的表达;高血压对照组Nox的基础活性是正常对照组的5倍.结论 高血压患者肾脏近曲小管细胞具有较高的Nox亚单位的活性,多巴胺受体和脂筏对Nox亚单位的抑制作用减弱.%Objective To investigate the expression and activity of NADPH oxidase ( Nox ) subunit in hypertensive renal proximal tubule cells ( HT ) and the regulatory role of dopamine receptors and lipid boat. Methods Cells were seperated into normotensive group( NT ) and hypertensive group ( HT ), and their respective control group was established by learing the cells intact. Glucose concentration gradient was used to extract cell membrane lipid rafts and non-lipid rafts region. The expression levels of Nox subunits were detected by Western blot, and NADPH oxidase activity were measured by Lucigenin Chemiluminescence. Results Compared with control group, dopamine receptor agonist of fenoldopam significantly reduced gp91 expression in membrane lipid raft regions both in NT[ (17 ±3

  18. Apheresis techniques for collection of peripheral blood progenitor cells.

    Science.gov (United States)

    Moog, Rainer

    2004-12-01

    The combination of effective mobilisation protocols and efficient use of apheresis machines has caused peripheral blood progenitor cells (PBPC) transplantation to grow rapidly. The development of apheresis technology has improved over the years. Today PBSC procedures have changed towards systems to minimise operator interaction and to reduce the collection of undesired cells such as polymorphonuclear cells and platelets using functionally closed, sterile environments for PBSC collection in keeping with Good Manufacturing Practice guidelines. Blood cell separators with continuous flow technique allow the processing of more blood than intermittent flow devices resulting in higher PBSC yields. Large volume leukapheresis with the processing of 3-4-fold donor's/patient's blood volume can increase the number of collected progenitor cells. Therefore, intermittent flow cell separators are indicated if only single vein access is available. Anticoagulant induced hypocalcaemia is an often observed side effect in long lasting PBPC harvesting and monitoring of electrolytes should be performed especially at the end of the apheresis procedure to supplement low levels of potassium, calcium or magnesium. Refinement and improvement of collection techniques continue to add to the armamentarium of current approaches for cancer and non-malignant conditions and will enable future strategies.

  19. cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

    Energy Technology Data Exchange (ETDEWEB)

    Gapstur, S.M.; Homma, S.; Dousa, T.P.

    1988-08-01

    Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-(32P)-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-(32P)cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-(32P)cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.

  20. Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules

    DEFF Research Database (Denmark)

    van Weering, Jan R.T.; Sessions, Richard B.; Traer, Colin J.

    2012-01-01

    -BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX-BARs display a restricted pattern of BAR domain-mediated dimerization, and by resolving a 2.8 Å structure of a SNX1-BAR domain homodimer, establish...... that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip...

  1. CRIP Homologues Maintain Apical Cytoskeleton to Regulate Tubule Size in C. elegans

    OpenAIRE

    Tong, Xiangyan; Buechner, Matthew

    2008-01-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of C. elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 sho...

  2. Intraluminal colonization into the seminiferous tubules in mice

    Directory of Open Access Journals (Sweden)

    Luis Guzmán-Masias

    2011-05-01

    Full Text Available Using the primordial germ cells transplant technique, we could be able preserve and multiply pluripotent cells in the receptor for a long period of time. In this work, We aim to evaluate intraluminal colonization of a cellular gonocyte suspension from 14.5 dpc fetus. Cellular suspension with PGC's were isolated from fetus male mice by two enzymatic digestion steps, and cellular suspensions were transplanted into the rete testis of the receptor animals that were previously injected with Busulfan to decrease their own spermatogenesis. In this research the intraluminal colonization was identified in 13.27%, demonstrating that transplantation of a cellular suspension from gonocytes of fetus of 14.5 dpc containing PGCs can colonize the seminiferous tubules and support the spermatogenesis.

  3. Topological defects control collective dynamics in neural progenitor cell cultures

    Science.gov (United States)

    Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

    2017-04-01

    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

  4. Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6

    OpenAIRE

    Hidalgo-Carcedo, Cristina; Hooper, Steven; Chaudhry, Shahid I.; Williamson, Peter; Harrington, Kevin; Leitinger, Birgit; Sahai, Erik

    2010-01-01

    Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell-cell junctions. Here we show that collective cancer cell invasion depends on reducing actomyosin contractility at sites of cell-cell contact. When actomyosin is not down-regulated at cell-cell contacts migrating cells lose cohesion. We provide a novel molecular mechanism for this down-regulation. Depletion of Discoidin Domain Receptor 1 (DDR1) blocks collective cancer cell i...

  5. Wild boars (Sus scrofa scrofa seminiferous tubules morphometry

    Directory of Open Access Journals (Sweden)

    Deiler Sampaio Costa

    2006-09-01

    Full Text Available The aim of this data was to analyze morphology and function of the seminiferous tubule in adult wild boars. Testes removed by unilateral castration of five animals were used. The testicular parenchyma was composed by 82.1±2.2% of seminiferous tubule and 17.9±2.2% of intertubular tissue. The tubular diameter was 249.2±33.0 µm and the seminiferous tubule lenght per gram of testis was 19.3±4.9m. The spermatogonial mitoses efficiency coefficient, meiotic index and spermatogenesis efficiency were 10.34, 2.71 and 30.5 respectively. Each Sertoli cell supported about 13 germinatives cells. The hystometric parameters studied were very similar to those related for domestic boars, however, the wild boars intrinsic efficiency of spermatogenesis and Sertoli cells indexes were smaller than in domestic boars.Objetivou-se com esta pesquisa estudar as características morfométricas e funcionais dos túbulos seminíferos de javalis adultos. Utilizaram-se testículos de cinco animais submetidos a orquiectomia unilateral. O parênquima testicular foi composto por 82,1 ± 2,2% de túbulos seminíferos e 17,9 ± 2,2% de tecido intertubular. O diâmetro tubular foi de 249,2 ± 33,0µm e o comprimento dos túbulos seminíferos por grama de testículo foi de 19,3 ± 4,9m. O coeficiente de eficiência das mitoses espermatogônias, o rendimento meiótico e o rendimento geral da espermatogênese foram, respectivamente, 10,34, 2,71 e 30,50. Cada célula de Sértoli suportou cerca de 13 células germinativas. Conclui-se que os parâmetros histométricos estudados nesta pesquisa foram muito semelhantes aos valores relatados para suínos domésticos, entretanto, o rendimento intrínseco da espermatogênese e os índices de células de Sértoli de javalis foram relativamente baixos quando comparados com aqueles animais.

  6. Interdigitated back contact solar cell with high-current collection

    Energy Technology Data Exchange (ETDEWEB)

    Garner, C. M.; Nasby, R. D.; Sexton, F. W.; Rodriguez, J. L.; Norwood, D. P.

    1981-01-01

    Internal current-collection efficiencies greater than 90 percent and energy-conversion efficiencies of 18 percent at 30 suns have been measured on a laboratory version of the interdigitated back contact (IBC) solar cell. The quantum efficiency at 600 nm was greater than 90 percent which implies a minority carrier lifetime of greater than 350 ..mu..sec and a front surface recombination velocity of less than 30 cm/sec on the better devices. To achieve these high-current collection efficiencies, a phosphorous gettering diffusion was performed on the front surface and then etched off. Also, thermal oxides were grown on the front and back of the cell to passivate the silicon surfaces. Although the internal collection efficiencies of the cell were high, series resistance caused the fill factor (FF) to decrease at concentrations above 30 suns. Dark current measurements on cells with a new grid spacing indicate that the series resistance is much lower than in the previous cell design. This should result in higher efficiencies at high concentration.

  7. Collective cell motility promotes chemotactic prowess and resistance to chemorepulsion.

    Science.gov (United States)

    Malet-Engra, Gema; Yu, Weimiao; Oldani, Amanda; Rey-Barroso, Javier; Gov, Nir S; Scita, Giorgio; Dupré, Loïc

    2015-01-19

    Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates. Whether malignant lymphocytes are capable of coordinating their motility in the context of chemokine gradients is, however, unknown. Here, we show that, upon exposure to CCL19 or CXCL12 gradients, malignant B and T lymphocytes assemble into clusters that migrate directionally and display a wider chemotactic sensitivity than individual cells. Physical modeling recapitulates cluster motility statistics and shows that intracluster cell cohesion results in noise reduction and enhanced directionality. Quantitative image analysis reveals that cluster migration runs are periodically interrupted by transitory rotation and random phases that favor leader cell turnover. Additionally, internalization of CCR7 in leader cells is accompanied by protrusion retraction, loss of polarity, and the ensuing replacement by new leader cells. These mechanisms ensure sustained forward migration and resistance to chemorepulsion, a behavior of individual cells exposed to steep CCL19 gradients that depends on CCR7 endocytosis. Thus, coordinated cluster dynamics confer distinct chemotactic properties, highlighting unexpected features of lymphoid cell migration.

  8. Effect of dental materials on gluconeogenesis in rat kidney tubules

    Energy Technology Data Exchange (ETDEWEB)

    Reichl, F.X.; Durner, J.; Mueckter, H.; Elsenhans, B.; Forth, W. [Muenchen Univ. (Germany). Walter-Straub-Institut fuer Pharmakologie und Toxikologie; Kunzelmann, K.H.; Hickel, R. [Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich (Germany); Spahl, W. [Institute of Organic Chemistry, Ludwig-Maximilians-University of Munich (Germany); Hume, W.R. [Dental Research Institute, Univ. of California, Los Angeles, CA (United States); Moes, G.W. [TNO Prins-Maurits-Laboratorium, Rijswijk (Netherlands)

    1999-09-01

    The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl{sub 2}) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules. From starved rats kidney tubules were prepared and isolated by digestion with collagenase. Every 10 min up to 60 min 1-ml samples were drawn from the cell suspension for quantitating the glucose content. Glucose formation in controls was 3.3 {+-} 0.2 nmol/mg . per min (mean {+-} SEM, n=21). Relative rates of glucose formation were obtained by expressing individual rates as a percentage of the corresponding control. X-Y concentration curves (effective concentration, EC) of the substances were calculated by fitting a four-parametric sigmoid function to the relative rates of glucose formation at various test concentrations. At the end of the incubation period cell viability was assessed by trypan blue exclusion. Cell viability decreased within the 60 min interval from 90 to approx. 80% (controls), <25 (HEMA), <20 (TEGDMA), <10 (MeHgCl), and <10% (HgCl{sub 2}). Values of 50% effective concentration (EC{sub 50}) were calculated from fitted curves. EC{sub 50} values were (mmol; mean {+-} SEM; n=4): HEMA, 17.7 {+-} 2.9; TEGDMA, 1.8 {+-} 0.2; MeHgCl, 0.018 {+-} 0.0005; and HgCl{sub 2}, 0.0016 {+-} 0.0005. The toxic effect of HgCl{sub 2} was {proportional{underscore}to}1000 or 10 000 higher than that of the dental composite components TEGDMA or HEMA, respectively. (orig.)

  9. Excretion of alkaloids by malpighian tubules of insects.

    Science.gov (United States)

    Maddrell, S H; Gardiner, B O

    1976-04-01

    Nicotine is transported at high rates by Malpighian tubules of larvae of Manduca sexta, Pieris brassicae and Rhodnius prolixus and the transport persists in the absence of alkaloid from the diet. In the fluid-secreting portion of Rhodnius tubules this transport is not coupled to ion transport, nor is it dependent on the physiological state of the animal. The transport, which can occur against a steep electrochemical gradient, shows saturation kinetics with a maximal rate of 700 pmol. min-1 per tubule and is half saturated at 2-3 mM. Nicotine transport independent of ion movements also occurs in the lower resorptive parts of Rhodnius tubules. Both portions of Rhodnius tubules can transport morphine and atropine. These alkaloids and nicotine compete with one naother and are presumed to be carried by the smae transport system. Nicotine transport in Rhodnius was unaffected by organic anions, such as amaranth and benzyl penicillin, or by the organic anion transport inhibitor, probenecid. Fluid secretion in 5-HT-stimulated tubules was reduced by atropine and nicotine, probably by blocking the 5-HT receptors. The Malpighian tubules of adult Calliphora erythrocephala and Musca domestica remove nicotine from bathing solutions, an unknown metabolic accumulating in the tubules. Adult P. brassicae and M. sexta do not exhibit transport of nicotine by their Malpighian tubules.

  10. Nonlinear analysis of lipid tubules by nonlocal beam model.

    Science.gov (United States)

    Shen, Hui-Shen

    2011-05-07

    Postbuckling, nonlinear bending and nonlinear vibration analyses are presented for lipid tubules. The lipid tubule is modeled as a nonlocal micro/nano-beam which contains small scale effect. The material properties are assumed to be size-dependent. The governing equation is solved by a two-step perturbation technique. The numerical results reveal that the small scale parameter e₀a reduces the postbuckling equilibrium paths, the static large deflections and natural frequencies of lipid tubules. In contrast, it increases the nonlinear to linear frequency ratios slightly for the lipid tubule with immovable end conditions.

  11. The pelvic kidney of male Ambystoma maculatum (Amphibia, urodela, ambystomatidae) with special reference to the sexual collecting ducts.

    Science.gov (United States)

    Siegel, Dustin S; Sever, David M; Aldridge, Robert D

    2010-12-01

    This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct

  12. Connecting single cell to collective cell behavior in a unified theoretical framework

    Science.gov (United States)

    George, Mishel; Bullo, Francesco; Campàs, Otger

    Collective cell behavior is an essential part of tissue and organ morphogenesis during embryonic development, as well as of various disease processes, such as cancer. In contrast to many in vitro studies of collective cell migration, most cases of in vivo collective cell migration involve rather small groups of cells, with large sheets of migrating cells being less common. The vast majority of theoretical descriptions of collective cell behavior focus on large numbers of cells, but fail to accurately capture the dynamics of small groups of cells. Here we introduce a low-dimensional theoretical description that successfully captures single cell migration, cell collisions, collective dynamics in small groups of cells, and force propagation during sheet expansion, all within a common theoretical framework. Our description is derived from first principles and also includes key phenomenological aspects of cell migration that control the dynamics of traction forces. Among other results, we explain the counter-intuitive observations that pairs of cells repel each other upon collision while they behave in a coordinated manner within larger clusters.

  13. Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.

    Science.gov (United States)

    Jayasinghe, Isuru D; Clowsley, Alexander H; Munro, Michelle; Hou, Yufeng; Crossman, David J; Soeller, Christian

    2015-01-07

    The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

  14. Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

    Directory of Open Access Journals (Sweden)

    Isuru D. Jayasinghe

    2014-12-01

    Full Text Available The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM, has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

  15. Collective cancer cell invasion induced by coordinated contractile stresses.

    Science.gov (United States)

    Jimenez Valencia, Angela M; Wu, Pei-Hsun; Yogurtcu, Osman N; Rao, Pranay; DiGiacomo, Josh; Godet, Inês; He, Lijuan; Lee, Meng-Horng; Gilkes, Daniele; Sun, Sean X; Wirtz, Denis

    2015-12-22

    The physical underpinnings of fibrosarcoma cell dissemination from a tumor in a surrounding collagen-rich matrix are poorly understood. Here we show that a tumor spheroid embedded in a 3D collagen matrix exerts large contractile forces on the matrix before invasion. Cell invasion is accompanied by complex spatially and temporally dependent patterns of cell migration within and at the surface of the spheroids that are fundamentally different from migratory patterns of individual fibrosarcoma cells homogeneously distributed in the same type of matrix. Cells display a continuous transition from a round morphology at the spheroid core, to highly aligned elongated morphology at the spheroid periphery, which depends on both β1-integrin-based cell-matrix adhesion and myosin II/ROCK-based cell contractility. This isotropic-to-anisotropic transition corresponds to a shift in migration, from a slow and unpolarized movement at the core, to a fast, polarized and persistent one at the periphery. Our results also show that the ensuing collective invasion of fibrosarcoma cells is induced by anisotropic contractile stresses exerted on the surrounding matrix.

  16. Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

    Science.gov (United States)

    Fujita, Naonobu; Huang, Wilson; Lin, Tzu-han; Groulx, Jean-Francois; Jean, Steve; Nguyen, Jen; Kuchitsu, Yoshihiko; Koyama-Honda, Ikuko; Mizushima, Noboru; Fukuda, Mitsunori; Kiger, Amy A

    2017-01-01

    Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms. DOI: http://dx.doi.org/10.7554/eLife.23367.001 PMID:28063257

  17. Interaction of the tracheal tubules of Scutigera coleoptrata (Chilopoda, Notostigmophora with glandular structures of the pericardial septum

    Directory of Open Access Journals (Sweden)

    Gero Hilken

    2015-06-01

    Full Text Available Notostigmophora (Scutigeromorpha exhibit a special tracheal system compared to other Chilopoda. The unpaired spiracles are localized medially on the long tergites and open into a wide atrium from which hundreds of tracheal tubules originate and extend into the pericardial sinus. Previous investigators reported that the tracheal tubules float freely in the hemolymph. However, here we show for the first time that the tracheal tubules are anchored to a part of the pericardial septum. Another novel finding is this part of the pericardial septum is structured as an aggregated gland on the basis of its specialized epithelium being formed by hundreds of oligocellular glands. It remains unclear whether the pericardial septum has a differently structure in areas that lack a connection with tracheal tubules. The tracheal tubules come into direct contact with the canal cells of the glands that presumably secrete mucous substances covering the entire luminal cuticle of the tracheal tubules. Connections between tracheae and glands have not been observed in any other arthropods.

  18. Akt recruits Dab2 to albumin endocytosis in the proximal tubule.

    Science.gov (United States)

    Koral, Kelly; Li, Hui; Ganesh, Nandita; Birnbaum, Morris J; Hallows, Kenneth R; Erkan, Elif

    2014-12-15

    Proximal tubule epithelial cells have a highly sophisticated endocytic machinery to retrieve the albumin in the glomerular filtrate. The megalin-cubilin complex and the endocytic adaptor disabled-2 (Dab2) play a pivotal role in albumin endocytosis. We previously demonstrated that protein kinase B (Akt) regulates albumin endocytosis in the proximal tubule through an interaction with Dab2. Here, we examined the nature of Akt-Dab2 interaction. The pleckstrin homology (PH) and catalytic domains (CD) of Akt interacted with the proline-rich domain (PRD) of Dab2 based on yeast-two hybrid (Y2H) experiments. Pull-down experiments utilizing the truncated constructs of Dab2 demonstrated that the initial 11 amino acids of Dab2-PRD were sufficient to mediate the interaction between Akt and Dab2. Endocytosis experiments utilizing Akt1- and Akt2-silencing RNA revealed that both Akt1 and Akt2 mediate albumin endocytosis in proximal tubule epithelial cells; therefore, Akt1 and Akt2 may play a compensatory role in albumin endocytosis. Furthermore, both Akt isoforms phosphorylated Dab2 at Ser residues 448 and 449. Ser-to-Ala mutations of these Dab2 residues inhibited albumin endocytosis and resulted in a shift in location of Dab2 from the peripheral to the perinuclear area, suggesting the physiological relevance of these phosphorylation sites in albumin endocytosis. We conclude that both Akt1 and Akt2 are involved in albumin endocytosis, and phosphorylation of Dab2 by Akt induces albumin endocytosis in proximal tubule epithelial cells. Further delineation of how Akt affects expression/phosphorylation of endocytic adaptors and receptors will enhance our understanding of the molecular network triggered by albumin overload in the proximal tubule.

  19. Single and collective cell migration: the mechanics of adhesions

    Science.gov (United States)

    De Pascalis, Chiara; Etienne-Manneville, Sandrine

    2017-01-01

    Chemical and physical properties of the environment control cell proliferation, differentiation, or apoptosis in the long term. However, to be able to move and migrate through a complex three-dimensional environment, cells must quickly adapt in the short term to the physical properties of their surroundings. Interactions with the extracellular matrix (ECM) occur through focal adhesions or hemidesmosomes via the engagement of integrins with fibrillar ECM proteins. Cells also interact with their neighbors, and this involves various types of intercellular adhesive structures such as tight junctions, cadherin-based adherens junctions, and desmosomes. Mechanobiology studies have shown that cell–ECM and cell–cell adhesions participate in mechanosensing to transduce mechanical cues into biochemical signals and conversely are responsible for the transmission of intracellular forces to the extracellular environment. As they migrate, cells use these adhesive structures to probe their surroundings, adapt their mechanical properties, and exert the appropriate forces required for their movements. The focus of this review is to give an overview of recent developments showing the bidirectional relationship between the physical properties of the environment and the cell mechanical responses during single and collective cell migration. PMID:28684609

  20. Malpighian Tubules as Novel Targets for Mosquito Control.

    Science.gov (United States)

    Piermarini, Peter M; Esquivel, Carlos J; Denton, Jerod S

    2017-01-24

    The Malpighian tubules and hindgut are the renal excretory tissues of mosquitoes; they are essential to maintaining hemolymph water and solute homeostasis. Moreover, they make important contributions to detoxifying metabolic wastes and xenobiotics in the hemolymph. We have focused on elucidating the molecular mechanisms of Malpighian tubule function in adult female mosquitoes and developing chemical tools as prototypes for next-generation mosquitocides that would act via a novel mechanism of action (i.e., renal failure). To date, we have targeted inward rectifier potassium (Kir) channels expressed in the Malpighian tubules of the yellow fever mosquito Aedes aegypti and malaria mosquito Anopheles gambiae. Inhibition of these channels with small molecules inhibits transepithelial K⁺ and fluid secretion in Malpighian tubules, leading to a disruption of hemolymph K⁺ and fluid homeostasis in adult female mosquitoes. In addition, we have used next-generation sequencing to characterize the transcriptome of Malpighian tubules in the Asian tiger mosquito Aedes albopictus, before and after blood meals, to reveal new molecular targets for potentially disrupting Malpighian tubule function. Within 24 h after a blood meal, the Malpighian tubules enhance the mRNA expression of genes encoding mechanisms involved with the detoxification of metabolic wastes produced during blood digestion (e.g., heme, NH₃, reactive oxygen species). The development of chemical tools targeting these molecular mechanisms in Malpighian tubules may offer a promising avenue for the development of mosquitocides that are highly-selective against hematophagous females, which are the only life stage that transmits pathogens.

  1. Effect of dental materials on gluconeogenesis in rat kidney tubules

    NARCIS (Netherlands)

    Reichl, F.X.; Durner, J.; Mückter, H.; Elsenhans, B.; Forth, W.; Kunzelmann, K.H.; Hickel, R.; Spahl, W.; Hume, W.R.; Moes, G.W.

    1999-01-01

    The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules. From starved rats kidney tubules were pr

  2. Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

    Science.gov (United States)

    Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

    2016-07-07

    The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing.

  3. Collective cell migration of thyroid carcinoma cells: a beneficial ability to override unfavourable substrates.

    Science.gov (United States)

    Lobastova, Liudmila; Kraus, Dominik; Glassmann, Alexander; Khan, Dilaware; Steinhäuser, Christian; Wolff, Christina; Veit, Nadine; Winter, Jochen; Probstmeier, Rainer

    2017-02-01

    Tumor cell invasion and metastasis are life threatening events. Invasive tumor cells tend to migrate as collective sheets. In the present in vitro study we aimed to (i) assess whether collective tumor cells gain benefits in their migratory potential compared to single cells and (ii) to identify its putative underlying molecular mechanisms. The migratory potential of single and collective carcinoma cells was assessed using video time lapse microscopy and cell migration assays in the absence and presence of seven potential gap junction inhibitors or the Rac1 inhibitor Z62954982. The perturbation of gap junctions was assessed using a dye diffusion assay. In addition, LDH-based cytotoxicity and RT-PCR-based expression analyses were performed. Whereas single breast, cervix and thyroid carcinoma cells were virtually immobile on unfavourable plastic surfaces, we found that they gained pronounced migratory capacities as collectives under comparable conditions. Thyroid carcinoma cells, that were studied in more detail, were found to express specific subsets of connexins and to form active gap junctions as revealed by dye diffusion analysis. Although all potential gap junction blockers suppressed intercellular dye diffusion in at least one of the cell lines tested, only two of them were found to inhibit collective cell migration and none of them to inhibit single cell migration. In the presence of the Rac1 inhibitor Z62954982 collective migration, but not single cell migration, was found to be reduced up to 20 %. Our data indicate that collective migration enables tumor cells to cross otherwise unfavourable substrate areas. This capacity seems to be independent of intercellular communication via gap junctions, whereas Rac1-dependent intracellular signalling seems to be essential.

  4. In vitro disinfection of dentinal tubules by various endodontics irrigants.

    Science.gov (United States)

    Buck, R; Eleazer, P D; Staat, R H

    1999-12-01

    Effectiveness of endodontic irrigants within dentinal tubules of human teeth was evaluated. Mid-sections of single-rooted teeth were prepared into dentin wedges. The pulpal sides of the sections were exposed to Micrococcus luteus or Bacillus megaterium that grew into the tubules. Irrigants used in the study included: 0.525% NaOCl, 0.12% chlorhexidine, RC Prep, 0.5% betadine iodine, and sterile H2O (as a control). Pulpal surfaces were exposed to an irrigant and then rinsed in sterile water. The samples were then cracked, exposing a fresh surface. Culture of the exposed dentin surfaces showed that selected irrigants reached to the far ends of the dentinal tubules in a concentration sufficient to kill 100% of the M. luteus. However B. megaterium was neither killed nor apparently inhibited by any irrigant. We conclude that endodontic irrigants permeate throughout dentinal tubules, but their effectiveness is dependent on the type of bacteria found within the tubules.

  5. The early history of tubulation in nerve repair.

    Science.gov (United States)

    IJpma, F F A; Van De Graaf, R C; Meek, M F

    2008-10-01

    The first experiments for bridging peripheral nerve gaps using nerve tubulation emerged in the 19th century. Because Gluck (1853-1942) is said to have performed the first animal experiment of nerve tubulation in 1880, it is interesting to explore the background and veracity of this claim. The original documents on nerve tubulation in the 19th century were studied. We conclude that the conduit that was initially used for nerve tubulation was derived from a resorbable decalcified bone tube developed for wound drainage by Neuber (1850-1932) in 1879. Gluck proposed the use of the bone tube as a guided conduit for regenerating nerves in 1881 but stated briefly that his experiments failed because of scar formation. Vanlair (1839-1914) documented the first successful application of nerve tubulation using a bone tube to bridge a 3 cm sciatic nerve defect in a dog in 1882.

  6. Self-(Un)rolling Biopolymer Microstructures: Rings, Tubules, and Helical Tubules from the Same Material.

    Science.gov (United States)

    Ye, Chunhong; Nikolov, Svetoslav V; Calabrese, Rossella; Dindar, Amir; Alexeev, Alexander; Kippelen, Bernard; Kaplan, David L; Tsukruk, Vladimir V

    2015-07-13

    We have demonstrated the facile formation of reversible and fast self-rolling biopolymer microstructures from sandwiched active-passive, silk-on-silk materials. Both experimental and modeling results confirmed that the shape of individual sheets effectively controls biaxial stresses within these sheets, which can self-roll into distinct 3D structures including microscopic rings, tubules, and helical tubules. This is a unique example of tailoring self-rolled 3D geometries through shape design without changing the inner morphology of active bimorph biomaterials. In contrast to traditional organic-soluble synthetic materials, we utilized a biocompatible and biodegradable biopolymer that underwent a facile aqueous layer-by-layer (LbL) assembly process for the fabrication of 2D films. The resulting films can undergo reversible pH-triggered rolling/unrolling, with a variety of 3D structures forming from biopolymer structures that have identical morphology and composition.

  7. Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo.

    Science.gov (United States)

    Ran, Shujun; Gu, Shensheng; Wang, Jia; Zhu, Cailian; Liang, Jingping

    2015-08-21

    Enterococcus faecalis is the species most frequently isolated from failed endodontic treatments because it can survive under stress conditions imposed by root canal treatment. The objective of this study was to determine the ability of E. faecalis to invade dentine tubules under alkaline and energy-starvation stress and to explore the potential mechanisms. Roots from single-rooted human teeth were infected with E. faecalis under alkaline and energy-starvation stress conditions. After 4 wk of culture, samples were processed to establish the tubule-penetration distance. In addition, the hydrophobicity of E. faecalis cells under these conditions was analysed and the expression of genes involved in adhesion was quantified by real-time quantitative PCR. Culture of E. faecalis under alkaline and energy-starvation stress conditions resulted in a marked reduction of tubule-penetration distance, a significant increase in hydrophobicity of the bacterial surface, and marked down-regulation of most adhesin genes compared with E. faecalis cultured in tryptic soy broth. The results indicate that the dentine tubule invasion ability of E. faecalis was markedly decreased under alkaline and glucose-starvation stress conditions, possibly because of the increase of hydrophobicity and down-regulation of some adhesion genes.

  8. Glycogen accumulation in the pars recta of the proximal tubule in Fanconi syndrome.

    Science.gov (United States)

    Bendon, R W; Hug, G

    1986-01-01

    We reviewed the renal pathology in 10 cases of renal Fanconi syndrome. Five cases showed the Armanni-Ebstein lesion, i.e., clear glycogen-filled cells limited to the pars recta of the proximal tubules. The 5 cases included 2 siblings with a unique syndrome characterized by death in infancy, severe Fanconi syndrome, severe rickets, carnitine deficiency, and atrophy of the exocrine pancreas. Two other siblings had glycogen storage disease type XI. One of 4 cases of putative tyrosinemia had the lesion. The ultrastructure was studied in 2 cases. The Armanni-Ebstein lesion in these cases was morphologically indistinguishable from that seen in diabetic patients dying after prolonged hyperglycemia. Glycosuria is the only common factor in both diabetic hyperglycemia and the varied proximal tubular diseases studied. The mechanism of the glycogen accumulation in this short parts recta segment of the proximal renal tubule was further investigated by reviewing the renal histology in cases of glycogen storage disease types I, II, III, and VIII. None showed the Armanni-Ebstein lesion, but type I showed glycogen deposition throughout the proximal tubule. Thus, the Armanni-Ebstein lesion is not the result of an enzymatic deficiency for glycogen synthesis in the convoluted tubules.

  9. Ultrastructural changes in renal proximal tubules after tetraethyllead intoxication

    Energy Technology Data Exchange (ETDEWEB)

    Chang, L.W. (Univ. of Arkansas for Medical Sciences, Little Rock); Wade, P.R.; Reuhl, K.R.; Olson, M.J.

    1980-10-01

    Tetraethyllead (TEL) has been shown to be both an occupational and an environmental hazard to human health. The present study investigates pathological changes in the kidney as a result of TEL poisoning. Rabbits were injected (ip) with 100 to 200 mg TEL, and controls were injected with an equal volume of normal saline solution. Animals were sacrificed upon onset of toxic symptoms (hyperirritation, tremor, and convulsion). Animals were perfused with 2.5% glutaraldehyde. Tissue samples from the renal cortex were obtained for electron microscopy. Pathological changes were not remarkable at the light microscopic level; however, electron microscopic examination revealed marked cytological changes in the epithelial cells of the proximal tubules (PT) of animals treated with TEL. Enlargement of apical vacuoles and accumulation of lysosomes and microbodies were prominent findings in many PT epithelial cells. Many lysosomes appeared to be atypical in nature, displaying a high degree of pleomorphism in size, shape, and density. Giant lysosomes measuring 8 to 10 ..mu..m in diameter and crystalloid bodies within lysosomes were also observed. Configurational changes (increased convolution, branching, vesiculation, and degranulation) of the rough endoplasmic reticulum leading to the formation of honeycomb-like bodies were also found in many PT epithelial cells. The formation of the honeycomb-like bodies may represent a hyperplastic, hypoactive form of the rough endoplasmic reticulum and denotes a disruption of protein synthesis in these cells by TEL.

  10. Notch1-Dll4 signalling and mechanical force regulate leader cell formation during collective cell migration.

    Science.gov (United States)

    Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D; Wong, Pak Kin

    2015-03-13

    At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct 'leader' phenotype with characteristic morphology and motility. However, the factors driving the leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here we use single-cell gene expression analysis and computational modelling to show that the leader cell identity is dynamically regulated by Dll4 signalling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signalling to dynamically regulate the density of leader cells during collective cell migration.

  11. BIN1 localizes the L-type calcium channel to cardiac T-tubules.

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    Ting-Ting Hong

    2010-02-01

    Full Text Available The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2 to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient

  12. CRIP homologues maintain apical cytoskeleton to regulate tubule size in C. elegans.

    Science.gov (United States)

    Tong, Xiangyan; Buechner, Matthew

    2008-05-01

    Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of Caenorhabditis elegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface.

  13. Cell-alignment patterns in the collective migration of cells with polarized adhesion

    Science.gov (United States)

    Matsushita, Katsuyoshi

    2017-03-01

    Dictyostelium discoideum (Dd) utilizes inhomogeneities in the distribution of cell-cell adhesion molecules on cell membranes for collective cell migration. A simple example of an inhomogeneity is a front-side (leading-edge) polarization in the distribution at the early streaming stage. Experiments have shown that the polarized cell-cell adhesion induces side-by-side contact between cells [Beug et al., Nature (London) 274, 445 (1978), 10.1038/274445a0]. This result is counterintuitive, as one would expect cells to align front to front in contact with each other on the basis of front-side polarization. In this work, we theoretically examine whether front-side polarization induces side-by-side contact in collective cell migration. We construct a model for expressing cells with this polarization based on the two-dimensional cellular Potts model. By a numerical simulation with this model, we find cell-cell alignment wherein cells form lateral arrays with side-by-side contacts as observed in the experiments.

  14. Genetic ablation of AQP2 in the mouse connecting tubules results in a mild urinary concentrating defect

    DEFF Research Database (Denmark)

    Kortenoeven, Marleen; Pedersen, Nis Borbye; Miller, Lance

    Body water balance is regulated in the kidney via the vasopressin (AVP) regulated water channel aquaporin-2 (AQP2) expressed in the connecting tubule (CNT) and the collecting duct (CD). Although crucial for the urinary concentrating mechanism, the relative role of AQP2 in the CNT and CD are not f...

  15. Transport of a fluorescent cAMP analog in teleost proximal tubules.

    Science.gov (United States)

    Reichel, Valeska; Masereeuw, Rosalinde; van den Heuvel, Jeroen J M W; Miller, David S; Fricker, Gert

    2007-12-01

    Previous studies have shown that killifish (Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady-state luminal accumulation of fluo-cAMP was concentrative, specific, and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (PKC activator), all of which signal substantial reductions in cell-to-lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin, and this reduction was blocked by the PKA inhibitor H-89. Finally, in membrane vesicles from Spodoptera frugiperda (Sf9) cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell-to-lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.

  16. Reconstruction of mouse testicular cellular microenvironments in long-term seminiferous tubule culture.

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    Juho-Antti Mäkelä

    Full Text Available Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1 and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger, LIN28, Gpr125 (G protein-coupled receptor 125, CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2-4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to

  17. From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate.

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    Jordi van Gestel

    2015-04-01

    Full Text Available The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles" of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

  18. AQP5 is expressed in type-B intercalated cells in the collecting duct system of the rat, mouse and human kidney.

    Science.gov (United States)

    Procino, Giuseppe; Mastrofrancesco, Lisa; Sallustio, Fabio; Costantino, Vincenzo; Barbieri, Claudia; Pisani, Francesco; Schena, Francesco Paolo; Svelto, Maria; Valenti, Giovanna

    2011-01-01

    We screened human kidney-derived multipotent CD133+/CD24+ ARPCs for the possible expression of all 13 aquaporin isoforms cloned in humans. Interestingly, we found that ARPCs expressed both AQP5 mRNA and mature protein. This novel finding prompted us to investigate the presence of AQP5 in situ in kidney. We report here the novel finding that AQP5 is expressed in human, rat and mouse kidney at the apical membrane of type-B intercalated cells. AQP5 is expressed in the renal cortex and completely absent from the medulla. Immunocytochemical analysis using segment- and cell type-specific markers unambiguously indicated that AQP5 is expressed throughout the collecting system at the apical membrane of type-B intercalated cells, where it co-localizes with pendrin. No basolateral AQPs were detected in type-B intercalated cells, suggesting that AQP5 is unlikely to be involved in the net trans-epithelial water reabsorption occurring in the distal tubule. An intriguing hypothesis is that AQP5 may serve an osmosensor for the composition of the fluid coming from the thick ascending limb. Future studies will unravel the physiological role of AQP5 in the kidney. Copyright © 2011 S. Karger AG, Basel.

  19. Guidance signalling regulates leading edge behaviour during collective cell migration of cardiac cells in Drosophila.

    Science.gov (United States)

    Raza, Qanber; Jacobs, J Roger

    2016-11-15

    Collective cell migration is the coordinated movement of cells, which organize tissues during morphogenesis, repair and some cancers. The motile cell membrane of the advancing front in collective cell migration is termed the Leading Edge. The embryonic development of the vertebrate and Drosophila hearts are both characterized by the coordinated medial migration of a bilateral cluster of mesodermal cells. In Drosophila, the cardioblasts form cohesive bilateral rows that migrate collectively as a unit towards the dorsal midline to form the dorsal vessel. We have characterized the collective cell migration of cardioblasts as an in vivo quantitative model to study the behaviour of the Leading Edge. We investigated whether guidance signalling through Slit and Netrin pathways plays a role in cell migration during heart development. Through time-lapse imaging and quantitative assessment of migratory behaviour of the cardioblasts in loss-of-function mutants, we demonstrate that both Slit and Netrin mediated signals are autonomously and concomitantly required to maximize migration velocity, filopodial and lamellipodial activities. Additionally, we show that another Slit and Netrin receptor, Dscam1, the role of which during heart development was previously unknown, is required for both normal migration of cardioblasts and luminal expansion. Leading edge behaviour analysis revealed a dosage dependent genetic interaction between Slit and Netrin receptors suggesting that downstream signalling through these receptors converge on a common output that increases leading edge activity of the cardioblasts. Finally, we found that guidance signalling maintains the balance between epithelial and mesenchymal characteristics of the migrating cardioblasts.

  20. Collection of peripheral progenitor cells: a comparison between Amicus and Cobe-Spectra blood cell separators.

    Science.gov (United States)

    Adorno, Gaspare; Del Proposto, Gianpaolo; Palombi, Francesca; Bruno, Antonio; Ballatore, Giovanna; Postorino, Massimiliano; Tendas, Andrea; Del Poeta, Giovanni; Isacchi, Giancarlo; Amadori, Sergio

    2004-04-01

    The authors compared the efficiency of two different blood cell separators (Amicus and Cobe-Spectra) in collecting peripheral blood progenitor cells for autologous or homologous transplantation. A total number of 129 procedures were performed, 36 with Spectra, 93 with Amicus. There was no difference between Spectra and Amicus efficiencies for CD34+ cell collection (46.685% vs 46.235%; p=n.s) but the platelet efficiencies were 17.31% and 12.54% respectively (p=0.04) and, if autologous and allogeneic collections were considered separately, a marked difference resulted in allogeneic platelet efficiency between 6 Spectra and 23 Amicus procedures (26.83% vs 8.68%, p=0.0004). The authors were able to demonstrate that in 70 Amicus autologous collections there was a different platelet efficiency, if peripheral count was considered: 12 procedures performed with a platelet count > 100 x 10(9)/l had a very low efficiency (6.86%), but this value increased if platelet count lowered (13.02% if between 100 and 50 x 10(9)/l, 22.63% if between 50 and 0 x 10(9)/l, 23 and 35 procedures respectively). The study is preliminary and the number of collections is little, but the overall data suggest that Spectra (AutoPBSC, V 6.0) and Amicus separators have the same efficiency for collecting CD34+ cells while Amicus procedures have a very low platelet contamination, especially with donors.

  1. Planar cell polarity signaling in collective cell movements during morphogenesis and disease.

    Science.gov (United States)

    Muñoz-Soriano, Verónica; Belacortu, Yaiza; Paricio, Nuria

    2012-12-01

    Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.

  2. Reactive oxygen species and IRF1 stimulate IFNα production by proximal tubules during ischemic AKI

    Science.gov (United States)

    Winterberg, Pamela D.; Wang, Yanxia; Lin, Keng-Mean; Hartono, John R.; Nagami, Glenn T.; Zhou, Xin J.; Shelton, John M.; Richardson, James A.

    2013-01-01

    We previously reported that expression of the transcription factor interferon regulatory factor 1 (IRF1) is an early, critical maladaptive signal expressed by renal tubules during murine ischemic acute kidney injury (AKI). We now show that IRF1 mediates signals from reactive oxygen species (ROS) generated during ischemic AKI and that these signals ultimately result in production of α-subtypes of type I interferons (IFNαs). We found that genetic knockout of the common type I IFN receptor (IFNARI−/−) improved kidney function and histology during AKI. There are major differences in the spatial-temporal production of the two major IFN subtypes, IFNβ and IFNαs: IFNβ expression peaks at 4 h, earlier than IFNαs, and continues at the same level at 24 h; expression of IFNαs also increases at 4 h but continues to increase through 24 h. The magnitude of the increase in IFNαs relative to baseline is much greater than that of IFNβ. We show by immunohistology and study of isolated cells that IFNβ is produced by renal leukocytes and IFNαs are produced by renal tubules. IRF1, IFNαs, and IFNARI were found on the same renal tubules during ischemic AKI. Furthermore, we found that ROS induced IFNα expression by renal tubules in vitro. This expression was inhibited by small interfering RNA knockdown of IRF1. Overexpression of IRF1 resulted in the production of IFNαs. Furthermore, we found that IFNα stimulated production of maladaptive proinflammatory CXCL2 by renal tubular cells. Altogether our data support the following autocrine pathway in renal tubular cells: ROS > IRF1 > IFNα > IFNARI > CXCL2. PMID:23657854

  3. Cubilin is essential for albumin reabsorption in the renal proximal tubule.

    Science.gov (United States)

    Amsellem, Sabine; Gburek, Jakub; Hamard, Ghislaine; Nielsen, Rikke; Willnow, Thomas E; Devuyst, Olivier; Nexo, Ebba; Verroust, Pierre J; Christensen, Erik I; Kozyraki, Renata

    2010-11-01

    Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.

  4. Assignment of the human gene for the water channel of renal collecting duct Aquaporin 2 (AQP2) to chromosome 12 region q12-->q13

    NARCIS (Netherlands)

    Deen, P M; Weghuis, D O; Sinke, R J; Geurts van Kessel, A; Wieringa, B; van Os, C H

    1994-01-01

    The chromosomal localization of the gene encoding Aquaporin 2 (previously called WCH-CD), which acts as a water channel in the collecting tubules of the kidney, was determined. Southern blot hybridizations of chromosomal DNA from a panel of 25 different human-rodent hybrid cell lines assigned AQP2 t

  5. Vasopressin-regulated miRNAs and AQP2-targeting miRNAs in kidney collecting duct cells.

    Science.gov (United States)

    Kim, Jae-Eun; Jung, Hyun Jun; Lee, Yu-Jung; Kwon, Tae-Hwan

    2015-04-01

    Mature microRNA (miRNA) acts as an important posttranscriptional regulator. We aimed to profile vasopressin-responsive miRNAs in kidney inner medullary collecting duct cells and to identify aquaporin-2 (AQP2)-targeting miRNAs. Microarray chip assay was carried out in inner medullary collecting duct tubule suspensions from rat kidneys in the absence or presence of desmopressin (dDAVP) stimulation (10(-9) M, 2 h). The results demonstrated 19 miRNAs, including both precursor and mature miRNAs, as potential candidates that showed significant changes in expression after dDAVP stimulation (P 1.3-fold changes in expression on the microarray (miR-127, miR-1, miR-873, miR-16, miR-206, miR-678, miR-496, miR-298, and miR-463) were further examined by quantitative real-time PCR, and target genes of the selected miRNAs were predicted. Next, to identify AQP2-targeting miRNAs, in silico analysis was performed. Four miRNAs (miR-32, miR-137, miR-216a, and miR-216b) target the 3'-untranslated region of rat AQP2 mRNA. Target seed regions of miR-32 and miR-137 were also conserved in the 3'-untranslated region of mouse AQP2 mRNA. Quantitative real-time PCR and immunoblot analysis demonstrated that dDAVP-induced AQP2 expression was significantly attenuated in mpkCCDc14 cells when cells were transfected with miRNA mimics of miR-32 or miR-137. Moreover, luciferase reporter assay demonstrated a significant decrease of AQP2 translation in mpkCCDc14 cells transfected with miRNA mimics of miR-32 or miR-137. The present study provides novel insights into the regulation of AQP2 by RNA interference; however, vasopressin-regulated miRNAs did not include miR-32 or miR-137, indicating that the interaction of miRNAs with the AQP2 regulatory pathway requires further analysis. Copyright © 2015 the American Physiological Society.

  6. Bistable cell fate specification as a result of stochastic fluctuations and collective spatial cell behaviour.

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    Daniel Stockholm

    Full Text Available BACKGROUND: In culture, isogenic mammalian cells typically display enduring phenotypic heterogeneity that arises from fluctuations of gene expression and other intracellular processes. This diversity is not just simple noise but has biological relevance by generating plasticity. Noise driven plasticity was suggested to be a stem cell-specific feature. RESULTS: Here we show that the phenotypes of proliferating tissue progenitor cells such as primary mononuclear muscle cells can also spontaneously fluctuate between different states characterized by the either high or low expression of the muscle-specific cell surface molecule CD56 and by the corresponding high or low capacity to form myotubes. Although this capacity is a cell-intrinsic property, the cells switch their phenotype under the constraints imposed by the highly heterogeneous microenvironment created by their own collective movement. The resulting heterogeneous cell population is characterized by a dynamic equilibrium between "high CD56" and "low CD56" phenotype cells with distinct spatial distribution. Computer simulations reveal that this complex dynamic is consistent with a context-dependent noise driven bistable model where local microenvironment acts on the cellular state by encouraging the cell to fluctuate between the phenotypes until the low noise state is found. CONCLUSIONS: These observations suggest that phenotypic fluctuations may be a general feature of any non-terminally differentiated cell. The cellular microenvironment created by the cells themselves contributes actively and continuously to the generation of fluctuations depending on their phenotype. As a result, the cell phenotype is determined by the joint action of the cell-intrinsic fluctuations and by collective cell-to-cell interactions.

  7. Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia.

    Science.gov (United States)

    Verlander, Jill W; Chu, Diana; Lee, Hyun-Wook; Handlogten, Mary E; Weiner, I David

    2013-09-01

    Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetase's specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+ -free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.

  8. Heparan Sulfate Proteoglycans Regulate Fgf Signaling and Cell Polarity during Collective Cell Migration

    Directory of Open Access Journals (Sweden)

    Marina Venero Galanternik

    2015-01-01

    Full Text Available Collective cell migration is a highly regulated morphogenetic movement during embryonic development and cancer invasion that involves the precise orchestration and integration of cell-autonomous mechanisms and environmental signals. Coordinated lateral line primordium migration is controlled by the regulation of chemokine receptors via compartmentalized Wnt/β-catenin and fibroblast growth factor (Fgf signaling. Analysis of mutations in two exostosin glycosyltransferase genes (extl3 and ext2 revealed that loss of heparan sulfate (HS chains results in a failure of collective cell migration due to enhanced Fgf ligand diffusion and loss of Fgf signal transduction. Consequently, Wnt/β-catenin signaling is activated ectopically, resulting in the subsequent loss of the chemokine receptor cxcr7b. Disruption of HS proteoglycan (HSPG function induces extensive, random filopodia formation, demonstrating that HSPGs are involved in maintaining cell polarity in collectively migrating cells. The HSPGs themselves are regulated by the Wnt/β-catenin and Fgf pathways and thus are integral components of the regulatory network that coordinates collective cell migration with organ specification and morphogenesis.

  9. Modelling Malpighian tubule crystals within the predatory soil mite Pergamasus longicornis (Mesostigmata: Parasitidae).

    Science.gov (United States)

    Bowman, Clive E

    2017-05-01

    The occurrence of refractive crystals (aka guanine) is characterised in the Malpighian tubules of the free-living predatory parasitiform soil mite Pergamasus longicornis (Berlese) from a temporal series of histological sections during and after feeding on larval dipteran prey. The tubular system behaves as a single uniform entity during digestion. Malpighian mechanisms are not the 'concentrative' mechanism sought for the early stasis in gut size during the second later phase of prey feeding. Nor are Malpighian changes associated with the time of 'anal dabbing' during feeding. Peak gut expansion precedes peak Malpighian tubule guanine crystal occurrence in a hysteretic manner. There is no evidence of Malpighian tubule expansion by fluid alone. Crystals are not found during the slow phase of liquidised prey digestion. Malpighian tubules do not appear to be osmoregulatory. Malpighian guanine is only observed 48 h to 10 days after the commencement of feeding. Post digestion guanine crystal levels in the expanded Malpighian tubules are high-peaking as a pulse 5 days after the start of feeding (i.e. after the gut is void of food at 52.5 h). The half-life of guanine elimination from the tubules is 53 h. Evidence for a physiological input cascade is found-the effective half-life of guanine appearance in the Malpighian tubules being 7.8-16.7 h. Crystals are found present at all times in the lumen of the rectal vesicle and not anywhere else lumenally in the gut at all. No guanine was observed inside gut cells. There is no evidence for the storage in the rectal vesicle of a 'pulse' of Malpighian excretory products from a discrete 'pulse' of prey ingestion. A latent egestive common catabolic phase in the gut is inferred commencing 12.5 h after the start of feeding which may cause the rectal vesicle to expand due to the catabolism of current or previous meals. Malpighian tubules swell as the gut contracts in size over time post-prandially. There is evidence that at a

  10. Mitochondrial DNA deletion of proximal tubules is the result of itai-itai disease.

    Science.gov (United States)

    Takebayashi, Shigeo; Jimi, Shiro; Segawa, Masaru; Takaki, Aya

    2003-03-01

    The pathogenesis of itai-itai disease continues to be controversial, although cadmium (Cd) poisoning which arises via polluted water and rice in Japan is likely involved. Until recently, however, a well-defined animal model for Cd intoxication was not available. An animal model for itai-itai disease was produced in rats by low-dose Cd treatment, intraperitoneally for a period of 70-80 weeks. Osteomalacia followed the renal damage. A gene deletion in the mitochondrial DNA was found in the mitochondria of the proximal tubule cells of rats with chronic Cd intoxication, as was shown by the increased smaller PCR product seen by gel electrophoresis in one DNA region, where ATPase and cytochrome oxidase genes are located. However, the PCR product was different from that seen with a gene deletion associated with aging: del4834bp. Renal damage from Cd intoxication initially caused mitochondrial dysfunction indicated by the disturbance in reabsorption in the proximal tubules and decreased amounts of ATP, ATPase, and cytochrome oxidase with gradually progressing tubular proteinuria, and, finally, chronic renal failure with tubulointerstitial damage throughout the renal cortex. These gave rise to osteomalacia, subsequently. We concluded that in Cd poisoning, a mitochondrial gene deletion in the mitochondria of the proximal tubule cells was the primary event for the pathogenesis of osteomalacia in itai-itai disease.

  11. Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae).

    Science.gov (United States)

    Catae, Aline Fernanda; Roat, Thaisa Cristina; De Oliveira, Regiane Alves; Nocelli, Roberta Cornélio Ferreira; Malaspina, Osmar

    2014-04-01

    Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non-target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC₅₀ (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide.

  12. Cadmium transport by the gut and Malpighian tubules of Chironomus riparius

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, Erin M., E-mail: leonarem@mcmaster.ca [Department of Biology, McMaster University, Hamilton, ON, L8S 4K1 (Canada); Pierce, Laura M. [Department of Biology, McMaster University, Hamilton, ON, L8S 4K1 (Canada); Gillis, Patricia L. [Aquatic Ecosystem Protection Research Division, Environment Canada, Burlington, ON, L7R 4A6 (Canada); Wood, Chris M.; O' Donnell, Michael J. [Department of Biology, McMaster University, Hamilton, ON, L8S 4K1 (Canada)

    2009-05-05

    Many aquatic insects are very insensitive to cadmium in short-term laboratory studies. LC50 values for larvae of the midge Chironomus riparius are over 25,000 times the Criterion Maximum Concentration in the United States Environmental Protection Agency (U.S. EPA (2000)) species sensitivity distribution (SSD). Excretion or sequestration of cadmium may contribute to insensitivity and we have therefore examined cadmium transport by isolated guts and renal tissues of C. riparius larvae. Regional differences of Cd transport along the gut were identified using a Cd{sup 2+}-selective microelectrode in conjunction with the Scanning Ion-Selective Electrode Technique (SIET). Cd is transported into the anterior midgut (AMG) cells from the lumen and out of the cells into the hemolymph. The transport of Cd from the gut lumen to the hemolymph exposes other tissues such as the nervous system and muscles to Cd. The gut segments which remove Cd from the hemolymph at the highest rate are the posterior midgut (PMG) and the ileum. In addition, assays using an isolated Malpighian (renal) tubule preparation have shown that the Malpighian tubules (MT) both sequester and secrete Cd. For larvae bathed in 10 {mu}mol l{sup -1} Cd, the tubules can secrete the entire hemolymph burden of Cd in {approx}15 h.

  13. MG53 is dispensable for T-tubule maturation but critical for maintaining T-tubule integrity following cardiac stress.

    Science.gov (United States)

    Zhang, Caimei; Chen, Biyi; Wang, Yihui; Guo, Ang; Tang, Yiqun; Khataei, Tahsin; Shi, Yun; Kutschke, William J; Zimmerman, Kathy; Weiss, Robert M; Liu, Jie; Benson, Christopher J; Hong, Jiang; Ma, Jianjie; Song, Long-Sheng

    2017-08-16

    The cardiac transverse (T)-tubule membrane system is the safeguard for cardiac function and undergoes dramatic remodeling in response to cardiac stress. However, the mechanism by which cardiomyocytes repair damaged T-tubule network remains unclear. In the present study, we tested the hypothesis that MG53, a muscle-specific membrane repair protein, antagonizes T-tubule damage to protect against maladaptive remodeling and thereby loss of excitation-contraction coupling and cardiac function. Using MG53-knockout (MG53-KO) mice, we first established that deficiency of MG53 had no impact on maturation of the T-tubule network in developing hearts. Additionally, MG53 ablation did not influence T-tubule integrity in unstressed adult hearts as late as 10months of age. Following left ventricular pressure overload-induced cardiac stress, MG53 protein levels were increased by approximately three-fold in wild-type mice, indicating that pathological stress induces a significant upregulation of MG53. MG53-deficient mice had worsened T-tubule disruption and pronounced dysregulation of Ca(2+) handling properties, including decreased Ca(2+) transient amplitude and prolonged time to peak and decay. Moreover, MG53 deficiency exacerbated cardiac hypertrophy and dysfunction and decreased survival following cardiac stress. Our data suggest MG53 is not required for T-tubule development and maintenance in normal physiology. However, MG53 is essential to preserve T-tubule integrity and thereby Ca(2+) handling properties and cardiac function under pathological cardiac stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Local pH domains regulate NHE3-mediated Na+ reabsorption in the renal proximal tubule

    Science.gov (United States)

    Burford, James L.; McDonough, Alicia A.; Holstein-Rathlou, Niels-Henrik; Peti-Peterdi, Janos

    2014-01-01

    The proximal tubule Na+/H+ exchanger 3 (NHE3), located in the apical dense microvilli (brush border), plays a major role in the reabsorption of NaCl and water in the renal proximal tubule. In response to a rise in blood pressure NHE3 redistributes in the plane of the plasma membrane to the base of the brush border, where NHE3 activity is reduced. This NHE3 redistribution is assumed to provoke pressure natriuresis; however, it is unclear how NHE3 redistribution per se reduces NHE3 activity. To investigate if the distribution of NHE3 in the brush border can change the reabsorption rate, we constructed a spatiotemporal mathematical model of NHE3-mediated Na+ reabsorption across a proximal tubule cell and compared the model results with in vivo experiments in rats. The model predicts that when NHE3 is localized exclusively at the base of the brush border, it creates local pH microdomains that reduce NHE3 activity by >30%. We tested the model's prediction experimentally: the rat kidney cortex was loaded with the pH-sensitive fluorescent dye BCECF, and cells of the proximal tubule were imaged in vivo using confocal fluorescence microscopy before and after an increase of blood pressure by ∼50 mmHg. The experimental results supported the model by demonstrating that a rise of blood pressure induces the development of pH microdomains near the bottom of the brush border. These local changes in pH reduce NHE3 activity, which may explain the pressure natriuresis response to NHE3 redistribution. PMID:25298526

  15. Piecewise-Constant-Model-Based Interior Tomography Applied to Dentin Tubules

    Directory of Open Access Journals (Sweden)

    Peng He

    2013-01-01

    Full Text Available Dentin is a hierarchically structured biomineralized composite material, and dentin’s tubules are difficult to study in situ. Nano-CT provides the requisite resolution, but the field of view typically contains only a few tubules. Using a plate-like specimen allows reconstruction of a volume containing specific tubules from a number of truncated projections typically collected over an angular range of about 140°, which is practically accessible. Classical computed tomography (CT theory cannot exactly reconstruct an object only from truncated projections, needless to say a limited angular range. Recently, interior tomography was developed to reconstruct a region-of-interest (ROI from truncated data in a theoretically exact fashion via the total variation (TV minimization under the condition that the ROI is piecewise constant. In this paper, we employ a TV minimization interior tomography algorithm to reconstruct interior microstructures in dentin from truncated projections over a limited angular range. Compared to the filtered backprojection (FBP reconstruction, our reconstruction method reduces noise and suppresses artifacts. Volume rendering confirms the merits of our method in terms of preserving the interior microstructure of the dentin specimen.

  16. Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment.

    Science.gov (United States)

    Jang, Kyung-Jin; Mehr, Ali Poyan; Hamilton, Geraldine A; McPartlin, Lori A; Chung, Seyoon; Suh, Kahp-Yang; Ingber, Donald E

    2013-09-01

    Kidney toxicity is one of the most frequent adverse events reported during drug development. The lack of accurate predictive cell culture models and the unreliability of animal studies have created a need for better approaches to recapitulate kidney function in vitro. Here, we describe a microfluidic device lined by living human kidney epithelial cells exposed to fluidic flow that mimics key functions of the human kidney proximal tubule. Primary kidney epithelial cells isolated from human proximal tubule are cultured on the upper surface of an extracellular matrix-coated, porous, polyester membrane that splits the main channel of the device into two adjacent channels, thereby creating an apical 'luminal' channel and a basal 'interstitial' space. Exposure of the epithelial monolayer to an apical fluid shear stress (0.2 dyne cm(-2)) that mimics that found in living kidney tubules results in enhanced epithelial cell polarization and primary cilia formation compared to traditional Transwell culture systems. The cells also exhibited significantly greater albumin transport, glucose reabsorption, and brush border alkaline phosphatase activity. Importantly, cisplatin toxicity and Pgp efflux transporter activity measured on-chip more closely mimic the in vivo responses than results obtained with cells maintained under conventional culture conditions. While past studies have analyzed kidney tubular cells cultured under flow conditions in vitro, this is the first report of a toxicity study using primary human kidney proximal tubular epithelial cells in a microfluidic 'organ-on-a-chip' microdevice. The in vivo-like pathophysiology observed in this system suggests that it might serve as a useful tool for evaluating human-relevant renal toxicity in preclinical safety studies.

  17. Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells.

    Science.gov (United States)

    Reffay, M; Parrini, M C; Cochet-Escartin, O; Ladoux, B; Buguin, A; Coscoy, S; Amblard, F; Camonis, J; Silberzan, P

    2014-03-01

    The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

  18. Transport of Streptococcus pneumoniae capsular polysaccharide in MHC Class II tubules.

    Directory of Open Access Journals (Sweden)

    Tom Li Stephen

    2007-03-01

    Full Text Available Bacterial capsular polysaccharides are virulence factors and are considered T cell-independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+ T cells in a major histocompatibility complex (MHC class II-dependent manner. The mechanism of carbohydrate presentation to CD4(+ T cells is unknown. We show in live murine dendritic cells (DCs that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell-dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide-carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.

  19. Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis.

    Science.gov (United States)

    Khan, Shenaz; Abu Jawdeh, Bassam G; Goel, Monu; Schilling, William P; Parker, Mark D; Puchowicz, Michelle A; Yadav, Satya P; Harris, Raymond C; El-Meanawy, Ashraf; Hoshi, Malcolm; Shinlapawittayatorn, Krekwit; Deschênes, Isabelle; Ficker, Eckhard; Schelling, Jeffrey R

    2014-03-01

    Chronic kidney disease progression can be predicted based on the degree of tubular atrophy, which is the result of proximal tubule apoptosis. The Na+/H+ exchanger NHE1 regulates proximal tubule cell survival through interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but pathophysiologic triggers for NHE1 inactivation are unknown. Because glomerular injury permits proximal tubule luminal exposure and reabsorption of fatty acid/albumin complexes, we hypothesized that accumulation of amphipathic, long-chain acyl-CoA (LC-CoA) metabolites stimulates lipoapoptosis by competing with the structurally similar PI(4,5)P2 for NHE1 binding. Kidneys from mouse models of progressive, albuminuric kidney disease exhibited increased fatty acids, LC-CoAs, and caspase-2-dependent proximal tubule lipoapoptosis. LC-CoAs and the cytosolic domain of NHE1 directly interacted, with an affinity comparable to that of the PI(4,5)P2-NHE1 interaction, and competing LC-CoAs disrupted binding of the NHE1 cytosolic tail to PI(4,5)P2. Inhibition of LC-CoA catabolism reduced NHE1 activity and enhanced apoptosis, whereas inhibition of proximal tubule LC-CoA generation preserved NHE1 activity and protected against apoptosis. Our data indicate that albuminuria/lipiduria enhances lipotoxin delivery to the proximal tubule and accumulation of LC-CoAs contributes to tubular atrophy by severing the NHE1-PI(4,5)P2 interaction, thereby lowering the apoptotic threshold. Furthermore, these data suggest that NHE1 functions as a metabolic sensor for lipotoxicity.

  20. Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies

    Directory of Open Access Journals (Sweden)

    Beisson Janine

    2001-03-01

    Full Text Available Abstract Background A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. Results Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. Conclusions The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop.

  1. Mechanism for collective cell alignment in Myxococcus xanthus bacteria

    CERN Document Server

    Balagam, Rajesh

    2015-01-01

    M. xanthus cells self-organize into clusters, aligned cell groups, at various stages of its lifecycle. Formation of these clusters is crucial for complex dynamic multi-cellular behavior of these bacteria. However the mechanism underlying the cell alignment and clustering is not fully understood. Motivated by studies of clustering in self-propelled rods, we hypothesized that M. xanthus cells can align and form clusters through pure mechanical interactions among cells and between cells and substrate. We test this hypothesis using an agent-based simulation framework where each agent is based on biophysical model of individual M. xanthus cell. We show that model agents, under realistic cell flexibility values, can align and form cell clusters but only when periodic reversals of cell directions are suppressed. However, by extending our model to introduce observed ability of cells to lay and follow slime trails, we show that effective trail following leads to clusters in reversing cells. Furthermore, we conclude th...

  2. Mechanical cell-matrix feedback explains pairwise and collective endothelial cell behavior in vitro.

    Directory of Open Access Journals (Sweden)

    René F M van Oers

    2014-08-01

    Full Text Available In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a the contractile forces that endothelial cells exert on the ECM, (b the resulting strains in the extracellular matrix, and (c the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.

  3. Ultrastructural study of seminiferous tubules in the rat after prenatal irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Hatier, R.; Grignon, G.; Touati, F.

    1982-01-01

    Is the presence of germinal cells necessary for the Sertoli cells to acquire normal features. To respond to this question we have studied the development of the Sertoli cells in rats irradiated at the end of the foetal life. In the prenatal irradiated rats, the lumen of the seminiferous tubules appears later than in the control rats. The Sertoli cells show numerous flexuous apical processes, with central microtubule bundles. These processes regress progressively after the 40th day of life when the tubular lumen appears; numerous junctional complexes differentiate with the same structure as those of control animals. There are important dilatations of the intercellular spaces. The cytoplasmic organelles show a normal development up to the 40th day of life. After this period, the rough endoplasmic reticulum and the Golgi apparatus clearly regress while important dilatations appear in the smooth endoplasmic reticulum and persist in the adult animal. From the 35th day on, the basal lamina of the seminiferous tubules is irregular and multilayered. The differentiation of the Sertoli cells seems to be independent of the presence of germinal cells until the 40th day of life and presents several particularities; thereafter the Sertoli cells show signs of regression.

  4. Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance.

    Directory of Open Access Journals (Sweden)

    Jeannette A E Söderberg

    Full Text Available The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7 are known, some of which are produced in the brain, others in fat body or intestine. Here we show that DILP5 is expressed in principal cells of the renal tubules of Drosophila and affects survival at stress. Renal (Malpighian tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. We investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK and its receptor DTKR during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2 in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone.

  5. Self-organization of engineered epithelial tubules by differential cellular motility

    Energy Technology Data Exchange (ETDEWEB)

    Mori, Hidetoshi; Gjorevski, Nikolce; Inman, Jamie L; Bissell, Mina J; Nelson, Celeste M

    2009-02-04

    Patterning of developing tissues arises from a number of mechanisms, including cell shape change, cell proliferation, and cell sorting from differential cohesion or tension. Here, we reveal that differences in cell motility can also lead to cell sorting within tissues. Using mosaic engineered mammary epithelial tubules, we found that cells sorted depending on their expression level of the membrane-anchored collagenase matrix metalloproteinase (MMP)-14. These rearrangements were independent of the catalytic activity of MMP14 but absolutely required the hemopexin domain. We describe a signaling cascade downstream of MMP14 through Rho kinase that allows cells to sort within the model tissues. Cell speed and persistence time were enhanced by MMP14 expression, but only the latter motility parameter was required for sorting. These results indicate that differential directional persistence can give rise to patterns within model developing tissues.

  6. Self-organization of engineered epithelial tubules by differential cellular motility

    Science.gov (United States)

    Mori, Hidetoshi; Gjorevski, Nikolce; Inman, Jamie L.; Bissell, Mina J.; Nelson, Celeste M.

    2009-01-01

    Patterning of developing tissues arises from a number of mechanisms, including cell shape change, cell proliferation, and cell sorting from differential cohesion or tension. Here, we reveal that differences in cell motility can also lead to cell sorting within tissues. Using mosaic engineered mammary epithelial tubules, we found that cells sorted depending on their expression level of the membrane-anchored collagenase matrix metalloproteinase (MMP)-14. These rearrangements were independent of the catalytic activity of MMP14 but absolutely required the hemopexin domain. We describe a signaling cascade downstream of MMP14 through Rho kinase that allows cells to sort within the model tissues. Cell speed and persistence time were enhanced by MMP14 expression, but only the latter motility parameter was required for sorting. These results indicate that differential directional persistence can give rise to patterns within model developing tissues. PMID:19706461

  7. Collective motion of cells crawling on a substrate: roles of cell shape and contact inhibition

    CERN Document Server

    Schnyder, Simon Kaspar; Molina, John Jairo; Yamamoto, Ryoichi

    2016-01-01

    Contact inhibition plays a crucial role in the motility of cells, the process of wound healing, and the formation of tumors. By mimicking the mechanical motion of calls crawling on a substrate using a pseudopod, we constructed a minimal model for migrating cells which gives rise to contact inhibition of locomotion (CIL) naturally. The model cell consists of two disks, one in the front (a pseudopod) and the other one in the back (cell body), connected by a finitely extensible spring. Despite the simplicity of the model, the cells' collective behavior is highly nontrivial, depending on the shape of cells and whether CIL is enabled or not. Cells with a small front circle (i.e. a narrow pseudopod) form immobile colonies. In contrast, cells with a large front circle (i.e. such as a lamellipodium) exhibit coherent migration without any explicit alignment mechanism being present in the model. This suggests that crawling cells often exhibit broad fronts because it helps them avoid clustering. Upon increasing the dens...

  8. Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney.

    Science.gov (United States)

    Holthöfer, H

    1988-08-01

    The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization. The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.

  9. Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro.

    Science.gov (United States)

    Kolman, Pavel; Pica, Angelo; Carvou, Nicolas; Boyde, Alan; Cockcroft, Shamshad; Loesch, Andrew; Pizzey, Arnold; Simeoni, Mariadelina; Capasso, Giovambattista; Unwin, Robert J

    2009-05-01

    We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after approximately 140 min) into the right jugular vein, and the fluorescence signal (at 650-670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 +/- 0.26, mean +/- SE, n = 10), indicating a "priming," or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 +/- 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand beta2-glycoprotein.

  10. Betal-integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+ signalling

    DEFF Research Database (Denmark)

    Prætorius, Helle; Prætorius, Jeppe; Nielsen, Søren

    2004-01-01

    of proximal tubules and thick ascending limbs. Immunogold-electron microscopy confirmed the presence of β1-integrin on primary cilia of MDCK cells and rat collecting ducts. Intracellular Ca2+ levels, monitored by fluorescence microscopy on fluo 4-loaded MDCK cells, significantly increased on addition...

  11. Collective migration models: Dynamic monitoring of leader cells in migratory/invasive disease processes

    Science.gov (United States)

    Dean, Zachary Steven

    Leader cells are a fundamental biological process that have only been investigated since the early 2000s. These cells have often been observed emerging at the edge of an artificial wound in 2D epithelial cell collective invasion, created with either a mechanical scrape from a pipette tip or from the removal of a plastic, physical blocker. During migration, the moving cells maintain cell-cell contacts, an important quality of collective migration; the leader cells originate from either the first or the second row, they increase in size compared to other cells, and they establish ruffled lamellipodia. Recent studies in 3D have also shown that cells emerging from an invading collective group that also exhibit leader-like properties. Exactly how leader cells influence and interact with follower cells as well as other cells types during collective migration, however, is another matter, and is a subject of intense investigation between many different labs and researchers. The majority of leader cell research to date has involved epithelial cells, but as collective migration is implicated in many different pathogenic diseases, such as cancer and wound healing, a better understanding of leader cells in many cell types and environments will allow significant improvement to therapies and treatments for a wide variety of disease processes. In fact, more recent studies on collective migration and invasion have broadened the field to include other cell types, including mesenchymal cancer cells and fibroblasts. However, the proper technology for picking out dynamic, single cells within a moving and changing cell population over time has severely limited previous investigation into leader cell formation and influence over other cells. In line with these previous studies, we not only bring new technology capable of dynamically monitoring leader cell formation, but we propose that leader cell behavior is more than just an epithelial process, and that it is a critical physiological

  12. Fundamental Limits to Collective Concentration Sensing in Cell Populations

    Science.gov (United States)

    Fancher, Sean; Mugler, Andrew

    2017-02-01

    The precision of concentration sensing is improved when cells communicate. Here we derive the physical limits to concentration sensing for cells that communicate over short distances by directly exchanging small molecules (juxtacrine signaling), or over longer distances by secreting and sensing a diffusive messenger molecule (autocrine signaling). In the latter case, we find that the optimal cell spacing can be large, due to a trade-off between maintaining communication strength and reducing signal cross-correlations. This leads to the surprising result that sparsely packed communicating cells sense concentrations more precisely than densely packed communicating cells. We compare our results to data from a wide variety of communicating cell types.

  13. Salinity alters snakeskin and mesh transcript abundance and permeability in midgut and Malpighian tubules of larval mosquito, Aedes aegypti.

    Science.gov (United States)

    Jonusaite, Sima; Donini, Andrew; Kelly, Scott P

    2017-03-01

    This study examined the distribution and localization of the septate junction (SJ) proteins snakeskin (Ssk) and mesh in osmoregulatory organs of larval mosquito (Aedes aegypti), as well as their response to altered environmental salt levels. Ssk and mesh transcripts and immunoreactivity were detected in tissues of endodermal origin such as the midgut and Malpighian tubules of A. aegypti larvae, but not in ectodermally derived hindgut and anal papillae. Immunolocalization of Ssk and mesh in the midgut and Malpighian tubules indicated that both proteins are concentrated at regions of cell-cell contact between epithelial cells. Transcript abundance of ssk and mesh was higher in the midgut and Malpighian tubules of brackish water (BW, 30% SW) reared A. aegypti larvae when compared with freshwater (FW) reared animals. Therefore, [(3)H]polyethylene glycol (MW 400Da, PEG-400) flux was examined across isolated midgut and Malpighian tubule preparations as a measure of their paracellular permeability. It was found that PEG-400 flux was greater across the midgut of BW versus FW larvae while the Malpighian tubules of BW-reared larvae had reduced PEG-400 permeability in conjunction with increased Cl(-) secretion compared to FW animals. Taken together, data suggest that Ssk and mesh are found in smooth SJs (sSJs) of larval A. aegypti and that their abundance alters in association with changes in epithelial permeability when larvae reside in water of differing salt content. This latter observation suggests that Ssk and mesh play a role in the homeostatic control of salt and water balance in larval A. aegypti. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

    Science.gov (United States)

    Terryn, Sara; Tanaka, Karo; Lengelé, Jean-Philippe; Olinger, Eric; Dubois-Laforgue, Danièle; Garbay, Serge; Kozyraki, Renata; Van Der Smissen, Patrick; Christensen, Erik I; Courtoy, Pierre J; Bellanné-Chantelot, Christine; Timsit, José; Pontoglio, Marco; Devuyst, Olivier

    2016-05-01

    Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of β2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A.

  15. Convergent extension: using collective cell migration and cell intercalation to shape embryos.

    Science.gov (United States)

    Tada, Masazumi; Heisenberg, Carl-Philipp

    2012-11-01

    Body axis elongation represents a common and fundamental morphogenetic process in development. A key mechanism triggering body axis elongation without additional growth is convergent extension (CE), whereby a tissue undergoes simultaneous narrowing and extension. Both collective cell migration and cell intercalation are thought to drive CE and are used to different degrees in various species as they elongate their body axis. Here, we provide an overview of CE as a general strategy for body axis elongation and discuss conserved and divergent mechanisms underlying CE among different species.

  16. Mechanism for Collective Cell Alignment in Myxococcus xanthus Bacteria.

    Directory of Open Access Journals (Sweden)

    Rajesh Balagam

    2015-08-01

    Full Text Available Myxococcus xanthus cells self-organize into aligned groups, clusters, at various stages of their lifecycle. Formation of these clusters is crucial for the complex dynamic multi-cellular behavior of these bacteria. However, the mechanism underlying the cell alignment and clustering is not fully understood. Motivated by studies of clustering in self-propelled rods, we hypothesized that M. xanthus cells can align and form clusters through pure mechanical interactions among cells and between cells and substrate. We test this hypothesis using an agent-based simulation framework in which each agent is based on the biophysical model of an individual M. xanthus cell. We show that model agents, under realistic cell flexibility values, can align and form cell clusters but only when periodic reversals of cell directions are suppressed. However, by extending our model to introduce the observed ability of cells to deposit and follow slime trails, we show that effective trail-following leads to clusters in reversing cells. Furthermore, we conclude that mechanical cell alignment combined with slime-trail-following is sufficient to explain the distinct clustering behaviors observed for wild-type and non-reversing M. xanthus mutants in recent experiments. Our results are robust to variation in model parameters, match the experimentally observed trends and can be applied to understand surface motility patterns of other bacterial species.

  17. Microchannel-free collection and single-cell isolation of yeast cells in a suspension using liquid standing wave

    Science.gov (United States)

    Matsutani, Akihiro; Takada, Ayako

    2016-11-01

    We demonstrate a microchannel-free collection method at nodes of liquid standing waves by the vertical vibration of a suspension including yeast cells. The pattern formation of the collection of cells using standing waves in a suspension was investigated by varying the frequency and waveform of vibrations. The single-cell isolation of yeast cells was achieved using a microenclosure array set at the nodes. In addition, we succeeded in the microchannel-free collection of yeast cells in a suspension, where patterns were formed by tapping vibration. The proposed technique is very simple and we believe that it will be useful for single-cell analysis and investigation.

  18. The caspase 3 sensor Phiphilux G2D2 is activated non-specifically in S1 renal proximal tubules

    Science.gov (United States)

    Hato, Takashi; Sandoval, Ruben; Dagher, Pierre C

    2016-01-01

    Tubular cell apoptosis is a major phenotype of cell death in various forms of acute kidney injury. Quantifying apoptosis in fixed tissues is problematic because apoptosis evolves over time and dead cells are rapidly cleared by the phagocytic system. Phiphilux is a fluorescent probe that is activated specifically by caspase 3 and does not inhibit the subsequent activity of this effector caspase. It has been used successfully to quantify apoptosis in cell culture. Here we examined the feasibility of using Phiphilux to measure renal tubular apoptosis progression over time in live animals using intravital 2-photon microscopy. Our results show that Phiphilux can detect apoptosis in S2 tubules but is activated non-specifically in S1 tubules.

  19. [Fructose-1,6-bisphosphatase--marker of damage to proximal renal tubules].

    Science.gov (United States)

    Kepka, Alina; Szajda, Sławomir D; Zwierz, Krzysztof

    2008-02-01

    Pathological processes disturbing function of renal proximal tubules, increase activity of fructose-1,6-bisphosphatase (FBP-1) in urine. FBP-1 is cytosolic enzyme which occured mainly in cells of proximal renal tubules, and to small extent in cells of pars recta. After damage to the cell membrane FBP-1 is more rapidly excreted to the urine, than enzymes residing in other cell organelles. Fructose-1,6-bisphosphatase was isolated from rabbit muscle in 1943 by Gomori, and from spinach in 1958 by Racker i Schröder. Highest activity of FBP-1 was found in liver and kidneys, lesser in ileum, leucocytes, muscles and brain. Fructose-1,6-bisphosphatase is one of four key enzymes of gluconeogenesis performing synthesis of glucose from non sugar substrates. FBP-1 catalyses hydrolysis of fructose-1,6-bisphosphate in cytoplasm of the cell. There are many reports on properties and significance of FBP-1 in plant and animal tissues, but only few reports on activity of this enzyme in urine. Reason for little interest in determination of FBP-1 activity in urine, is relative instability of this enzyme in urine.

  20. Problems in biology with many scales of length: Cell-cell adhesion and cell jamming in collective cellular migration.

    Science.gov (United States)

    Pegoraro, Adrian F; Fredberg, Jeffrey J; Park, Jin-Ah

    2016-04-10

    As do all things in biology, cell mechanosensation, adhesion and migration begin at the scale of the molecule. Collections of molecules assemble to comprise microscale objects such as adhesions, organelles and cells. And collections of cells in turn assemble to comprise macroscale tissues. From the points of view of mechanism and causality, events at the molecular scale are seen most often as being the most upstream and, therefore, the most fundamental and the most important. In certain collective systems, by contrast, events at many scales of length conspire to make contributions of equal importance, and even interact directly and strongly across disparate scales. Here we highlight recent examples in cellular mechanosensing and collective cellular migration where physics at some scale bigger than the cell but smaller than the tissue - the mesoscale - becomes the missing link that is required to tie together findings that might otherwise seem counterintuitive or even unpredictable. These examples, taken together, establish that the phenotypes and the underlying physics of collective cellular migration are far richer than previously anticipated. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Collective cell traction force analysis on aligned smooth muscle cell sheet between three-dimensional microwalls.

    Science.gov (United States)

    Zhang, Ying; Ng, Soon Seng; Wang, Yilei; Feng, Huixing; Chen, Wei Ning; Chan-Park, Mary B; Li, Chuan; Chan, Vincent

    2014-04-06

    During the past two decades, novel biomaterial scaffold for cell attachment and culture has been developed for applications in tissue engineering, biosensing and regeneration medicine. Tissue engineering of blood vessels remains a challenge owing to the complex three-layer histology involved. In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of vascular smooth muscle cells (SMCs) inside the tunica media, which is known to be critical for vasoconstriction and vasodilation of the circulatory system. Until now, there has been a lack of understanding on the mechanotransduction of the SMC layer during the transformation from viable synthetic to quiescent contractile phenotypes. In this study, microfabricated arrays of discontinuous microwalls coated with fluorescence microbeads were developed to probe the mechanotransduction of the SMC layer. First, the system was exploited for stimulating the formation of a highly aligned orientation of SMCs in native tunica medium. Second, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of a polyacrylamide gel layer coated on the discontinuous microwall arrays. Third, the conventional traction force assay for single cell measurement was extended for applications in three-dimensional cell aggregates. Then, the biophysical effects of discontinuous microwalls on the mechanotransduction of the SMC layer undergoing cell alignment were probed. Generally, the cooperative multiple cell-cell and cell-microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite-element modelling. The results show that the traction forces of highly aligned cells lying in the middle region between two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of Von Mises stress during the cell alignment process were dependent on the collective cell

  2. The Tubulation Activity of a Fission Yeast F-BAR Protein Is Dispensable for Its Function in Cytokinesis

    Directory of Open Access Journals (Sweden)

    Nathan A. McDonald

    2016-01-01

    Full Text Available F-BAR proteins link cellular membranes to the actin cytoskeleton in many biological processes. Here we investigated the function of the Schizosaccharomyces pombe Imp2 F-BAR domain in cytokinesis and find that it is critical for Imp2’s role in contractile ring constriction and disassembly. To understand mechanistically how the F-BAR domain functions, we determined its structure, elucidated how it interacts with membranes, and identified an interaction between dimers that allows helical oligomerization and membrane tubulation. Using mutations that block either membrane binding or tubulation, we find that membrane binding is required for Imp2’s cytokinetic function but that oligomerization and tubulation, activities often deemed central to F-BAR protein function, are dispensable. Accordingly, F-BARs that do not have the capacity to tubulate membranes functionally substitute for the Imp2 F-BAR, establishing that its major role is as a cell-cycle-regulated bridge between the membrane and Imp2 protein partners, rather than as a driver of membrane curvature.

  3. The Tubulation Activity of a Fission Yeast F-BAR Protein Is Dispensable for Its Function in Cytokinesis.

    Science.gov (United States)

    McDonald, Nathan A; Takizawa, Yoshimasa; Feoktistova, Anna; Xu, Ping; Ohi, Melanie D; Vander Kooi, Craig W; Gould, Kathleen L

    2016-01-26

    F-BAR proteins link cellular membranes to the actin cytoskeleton in many biological processes. Here we investigated the function of the Schizosaccharomyces pombe Imp2 F-BAR domain in cytokinesis and find that it is critical for Imp2's role in contractile ring constriction and disassembly. To understand mechanistically how the F-BAR domain functions, we determined its structure, elucidated how it interacts with membranes, and identified an interaction between dimers that allows helical oligomerization and membrane tubulation. Using mutations that block either membrane binding or tubulation, we find that membrane binding is required for Imp2's cytokinetic function but that oligomerization and tubulation, activities often deemed central to F-BAR protein function, are dispensable. Accordingly, F-BARs that do not have the capacity to tubulate membranes functionally substitute for the Imp2 F-BAR, establishing that its major role is as a cell-cycle-regulated bridge between the membrane and Imp2 protein partners, rather than as a driver of membrane curvature.

  4. Collective Calcium Dynamics in Networks of Communicating Cells

    Science.gov (United States)

    Byrd, Tommy; Potter, Garrett; Sun, Bo; Mugler, Andrew

    Cells can sense and encode information about their environment with remarkable precision. These properties have been studied extensively for single cells, but intercellular communication is known to be important for both single- and multicellular organisms. Here, we examine calcium dynamics of fibroblast cells exposed to external ATP stimuli, and the effects of communication and stimulus strength on cells' response. Experimental results show that increasing communication strength induces a greater fraction of cells to exhibit oscillatory calcium dynamics, but the frequencies of oscillation do not systematically shift with ATP strength. We developed a model of calcium signaling by adding noise, communication, and cell-to-cell variability to the model of Tang and Othmer. This model reproduces cells' increased tendency to oscillate as a function of communication strength, and frequency encoding is nearly removed at the global level. Our model therefore suggests that the propensity of cells to oscillate, rather than frequency encoding, determines the response to external ATP. These results suggest that the system lies near a critical boundary separating non-oscillatory and oscillatory calcium dynamics.

  5. Fundamental limits to collective concentration sensing in cell populations

    CERN Document Server

    Fancher, Sean

    2016-01-01

    The precision of concentration sensing is improved when cells communicate. Here we derive the physical limits to concentration sensing for cells that communicate over short distances by directly exchanging small molecules (juxtacrine signaling), or over longer distances by secreting and sensing a diffusive messenger molecule (autocrine signaling). In the latter case, we find that the optimal cell spacing can be large, due to a tradeoff between maintaining communication strength and reducing signal cross-correlations. This leads to the surprising result that autocrine signaling allows more precise sensing than juxtacrine signaling for sufficiently large populations. We compare our results to data from a wide variety of communicating cell types.

  6. Natural history of seminiferous tubule degeneration in Klinefelter syndrome

    DEFF Research Database (Denmark)

    Aksglaede, Lise; Wikström, Anne M; Rajpert-De Meyts, Ewa

    2006-01-01

    Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates dramatic...

  7. Innervation of the renal proximal convoluted tubule of the rat

    Energy Technology Data Exchange (ETDEWEB)

    Barajas, L.; Powers, K. (Harbor-UCLA Medical Center, Torrance (USA))

    1989-12-01

    Experimental data suggest the proximal tubule as a major site of neurogenic influence on tubular function. The functional and anatomical axial heterogeneity of the proximal tubule prompted this study of the distribution of innervation sites along the early, mid, and late proximal convoluted tubule (PCT) of the rat. Serial section autoradiograms, with tritiated norepinephrine serving as a marker for monoaminergic nerves, were used in this study. Freehand clay models and graphic reconstructions of proximal tubules permitted a rough estimation of the location of the innervation sites along the PCT. In the subcapsular nephrons, the early PCT (first third) was devoid of innervation sites with most of the innervation occurring in the mid (middle third) and in the late (last third) PCT. Innervation sites were found in the early PCT in nephrons located deeper in the cortex. In juxtamedullary nephrons, innervation sites could be observed on the PCT as it left the glomerulus. This gradient of PCT innervation can be explained by the different tubulovascular relationships of nephrons at different levels of the cortex. The absence of innervation sites in the early PCT of subcapsular nephrons suggests that any influence of the renal nerves on the early PCT might be due to an effect of neurotransmitter released from renal nerves reaching the early PCT via the interstitium and/or capillaries.

  8. Natural history of seminiferous tubule degeneration in Klinefelter syndrome

    DEFF Research Database (Denmark)

    Aksglaede, Lise; Wikström, Anne M; Rajpert-De Meyts, Ewa

    2006-01-01

    Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates dramatic......Klinefelter syndrome (47,XXY) is characterized by small, firm testis, gynaecomastia, azoospermia and hypergonadotropic hypogonadism. Degeneration of the seminiferous tubules in 47,XXY males is a well-described phenomenon. It begins in the fetus, progresses through infancy and accelerates...... dramatically at the time of puberty with complete hyalinization of the seminiferous tubules, although a few tubules with spermatogenesis may be present in adult life. Activation of the pituitary-gonadal axis at 3 months of age is seen in Klinefelter boys similar to healthy boys. However, the level...... of testosterone in Klinefelter boys is significantly lower than in controls. After this 'minipuberty', the hormone levels decline to normal prepubertal levels until puberty. In puberty, an initial rise in testosterone, inhibin B, LH and FSH occurs in Klinefelter boys. However, the rise in testosterone levels off...

  9. Collecting Duct Renal Cell Carcinoma Found to Involve the Collecting System During Partial Nephrectomy: A Case Report

    Directory of Open Access Journals (Sweden)

    Andrew C Harbin

    2015-06-01

    Full Text Available Collecting duct carcinoma (CDC is a rare and aggressive form of renal cell carcinoma (RCC arising from the principal cells of the collecting duct.  One third of cases present with metastatic disease, but many present in a manner similar to conventional RCC or urothelial carcinoma (UC.  We discuss a case of CDC which presented as a small mass at the cortico-medullary junction, and was discovered at robotic partial nephrectomy (RPN to be grossly involving the collecting system. A 62-year-old man presented with a small renal mass suspicious for RCC, which was found on computed tomography (CT after an episode of gross hematuria.  After thorough workup, RPN was attempted; however, intraoperatively the mass was found to be involving the collecting system.  Radical nephroureterectomy was performed, and the pathology report revealed CDC.  CDC is a rare and aggressive form of RCC.  While many cases are metastatic at diagnosis, most patients present with the incidental finding of a small renal mass.  There are no reports of a CDC involving the collecting system at RPN after negative ureteroscopy preoperatively.  The adjuvant therapeutic options for CDC are limited, and long term survival is poor.    

  10. Na+-H+ exchanger-1 (NHE1) regulation in kidney proximal tubule.

    Science.gov (United States)

    Parker, Mark D; Myers, Evan J; Schelling, Jeffrey R

    2015-06-01

    The ubiquitously expressed plasma membrane Na(+)-H(+) exchanger NHE1 is a 12 transmembrane-spanning protein that directs important cell functions such as homeostatic intracellular volume and pH control. The 315 amino acid cytosolic tail of NHE1 binds plasma membrane phospholipids and multiple proteins that regulate additional, ion-translocation independent functions. This review focuses on NHE1 structure/function relationships, as well as the role of NHE1 in kidney proximal tubule functions, including pH regulation, vectorial Na(+) transport, cell volume control and cell survival. The implications of these functions are particularly critical in the setting of progressive, albuminuric kidney diseases, where the accumulation of reabsorbed fatty acids leads to disruption of NHE1-membrane phospholipid interactions and tubular atrophy, which is a poor prognostic factor for progression to end stage renal disease. This review amplifies the vital role of the proximal tubule NHE1 Na(+)-H(+) exchanger as a kidney cell survival factor.

  11. Dynamics of Cell Ensembles on Adhesive Micropatterns: Bridging the Gap between Single Cell Spreading and Collective Cell Migration.

    Directory of Open Access Journals (Sweden)

    Philipp J Albert

    2016-04-01

    Full Text Available The collective dynamics of multicellular systems arise from the interplay of a few fundamental elements: growth, division and apoptosis of single cells; their mechanical and adhesive interactions with neighboring cells and the extracellular matrix; and the tendency of polarized cells to move. Micropatterned substrates are increasingly used to dissect the relative roles of these fundamental processes and to control the resulting dynamics. Here we show that a unifying computational framework based on the cellular Potts model can describe the experimentally observed cell dynamics over all relevant length scales. For single cells, the model correctly predicts the statistical distribution of the orientation of the cell division axis as well as the final organisation of the two daughters on a large range of micropatterns, including those situations in which a stable configuration is not achieved and rotation ensues. Large ensembles migrating in heterogeneous environments form non-adhesive regions of inward-curved arcs like in epithelial bridge formation. Collective migration leads to swirl formation with variations in cell area as observed experimentally. In each case, we also use our model to predict cell dynamics on patterns that have not been studied before.

  12. The effect of exercise training on transverse tubules in normal, remodeled, and reverse remodeled hearts

    OpenAIRE

    Kemi, Ole J.; Hoydal, Morten A; MacQuaide, Niall; Haram, Per M; Koch, Lauren G.; Steven L Britton; Ellingsen, Oyvind; Smith, Godfrey L.; Wisloff, Ulrik

    2011-01-01

    The response of transverse (T)-tubules to exercise training in health and disease remains unclear. Therefore, we studied the effect of exercise training on the density and spacing of left ventricle cardiomyocyte T-tubules in normal and remodeled hearts that associate with detubulation, by confocal laser scanning microscopy. First, exercise training in normal rats increased cardiomyocyte volume by 16% (P < 0.01), with preserved T-tubule density. Thus, the T-tubules adapted to the physiologi...

  13. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

    Science.gov (United States)

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S.; Riahi, Reza; Wong, Pak Kin

    2016-03-01

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

  14. Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

    Science.gov (United States)

    Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S; Riahi, Reza; Wong, Pak Kin

    2016-03-03

    When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

  15. Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin.

    Science.gov (United States)

    Love, R M; McMillan, M D; Park, Y; Jenkinson, H F

    2000-03-01

    Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease. Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8. An isogenic noninvasive mutant of S. gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P. gingivalis. Heterologous expression of the S. mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant of P. gingivalis had 50% reduced binding to S. gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S. gordonii wild-type cells. Expression of the S. gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P. gingivalis, penetrate dentinal tubules, and promote P. gingivalis coinvasion of dentin. The results demonstrate that collagen-binding and P. gingivalis-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of P. gingivalis to coinvade dentinal tubules with S. gordonii but not with S. mutans. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology

  16. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

    Directory of Open Access Journals (Sweden)

    E.V. Seliverstova

    2015-04-01

    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  17. Acute Tubuler Necrosis Related to Rhabdomyolysis

    Directory of Open Access Journals (Sweden)

    Fatma Sarı DOĞAN

    2014-05-01

    Full Text Available Rhabdomyolysis is a clinical and laboratory syndrome due to traumatic or non-traumatic injury that leads muscle cell contents participation into circulation. Dehydration and acidosis may cause myoglobinuric acute renal failure in patient with rhabdomyolysis. This case presents a 27-year-old male referred to emergency unit with weakness and abdominal ache who has a story of urine decrease and trauma exposure. Diagnosis of rhabdomyolysis in this case highlights the importance of anamnesis in early diagnosis and treatment.

  18. NMR-based urine analysis in rats: prediction of proximal tubule kidney toxicity and phospholipidosis.

    Science.gov (United States)

    Lienemann, Kai; Plötz, Thomas; Pestel, Sabine

    2008-01-01

    The aim of safety pharmacology is early detection of compound-induced side-effects. NMR-based urine analysis followed by multivariate data analysis (metabonomics) identifies efficiently differences between toxic and non-toxic compounds; but in most cases multiple administrations of the test compound are necessary. We tested the feasibility of detecting proximal tubule kidney toxicity and phospholipidosis with metabonomics techniques after single compound administration as an early safety pharmacology approach. Rats were treated orally, intravenously, inhalatively or intraperitoneally with different test compounds. Urine was collected at 0-8 h and 8-24 h after compound administration, and (1)H NMR-patterns were recorded from the samples. Variation of post-processing and feature extraction methods led to different views on the data. Support Vector Machines were trained on these different data sets and then aggregated as experts in an Ensemble. Finally, validity was monitored with a cross-validation study using a training, validation, and test data set. Proximal tubule kidney toxicity could be predicted with reasonable total classification accuracy (85%), specificity (88%) and sensitivity (78%). In comparison to alternative histological studies, results were obtained quicker, compound need was reduced, and very importantly fewer animals were needed. In contrast, the induction of phospholipidosis by the test compounds could not be predicted using NMR-based urine analysis or the previously published biomarker PAG. NMR-based urine analysis was shown to effectively predict proximal tubule kidney toxicity after single compound administration in rats. Thus, this experimental design allows early detection of toxicity risks with relatively low amounts of compound in a reasonably short period of time.

  19. SCANNING ELECTRON MICROSCOPIC INVESTIGATION OF DENTINAL TUBULES IN MONKEY DENTIN SCANNING ELECTRON MICROSCOPIC INVESTIGATION OF DENTINAL TUBULES IN Cebus apella DENTIN

    Directory of Open Access Journals (Sweden)

    João Humberto Antoniazzi

    2009-12-01

    Full Text Available

    The aim of the study was to investigate the number and diameter of the Cebus apella dentinal tubules. The roots of the Cebus apella teeth were examined in specific tooth locations: the apical, middle and cervical dentin. The calculations were based on the scanning electron microscope photographs of the fractured surfaces. The results showed that the average number of dentinal tubules for each location was: 74,800 tubules/mm2 for apical root dentin, 90,000 tubules/mm2 for mid-root dentin, 91,600 tubules/mm2 for cervical root dentin. The average diameter was the following: apical root dentin, 4,30µm; mid-root dentin, 4,37µm; cervical root dentin,  5,23µm. These findings demonstrate that the Cebus apella teeth are a suitable substitute for human in endodontics studies. 

    KEY WORDS: Dentin, dentinal tubules, teeth.
    The aim of the study was to investigate the number and diameter of the Cebus apella dentinal tubules. The roots of the Cebus apella teeth were examined in specific tooth locations: the apical, middle and cervical dentin. The calculations were based on the scanning electron microscope photographs of the fractured surfaces. The results showed that the average number of dentinal tubules for each location was: 74,800 tubules/mm2 for apical root dentin, 90,000 tubules/mm2 for mid-root dentin, 91,600 tubules/mm2 for cervical root dentin. The average diameter was the following: apical root dentin, 4,30µm; mid-root dentin, 4,37µm; cervical root dentin,  5,23µm. These findings demonstrate that the Cebus apella teeth are a suitable substitute for human in endodontics studies. 

    KEY WORDS: Dentin, dentinal tubules, teeth.

  20. Use of laboratory tests to guide initiation of autologous hematopoietic progenitor cell collection by apheresis: results from the multicenter hematopoietic progenitor cell collection by Apheresis Laboratory Trigger Survey.

    Science.gov (United States)

    Makar, Robert S; Padmanabhan, Anand; Kim, Haewon C; Anderson, Christina; Sugrue, Michele W; Linenberger, Michael

    2014-10-01

    Limited literature describes the value of laboratory "triggers" to guide collection of peripheral blood (PB) hematopoietic progenitor cells (HPCs) by apheresis [HPC(A)]. We used a web-based survey to determine which parameters are used to initiate autologous HPC(A) collection in adult and pediatric patients and to identify common practice patterns. Members of the AABB Cellular Therapy Product Collection and Clinical Practices Subsection and the American Society for Apheresis HPC Donor Subcommittee drafted and developed relevant survey questions. A web link to the survey was distributed by electronic newsletter or email. Responses from 67 programs that perform autologous HPC(A) collections, including academic medical centers (n = 46), blood centers (n = 10), community hospitals (n = 5), and a variety of other medical institutions (n = 6), were analyzed. Ninety-three percent (62/67) of programs used a laboratory parameter to initiate HPC(A) collection. In both adult (40/54, 74%) and pediatric (29/38, 76%) patients, the PB CD34+ cell count was the most common parameter used to initiate HPC(A) collection. The median PB CD34+ trigger value was 10/μL for both patient populations. Among centers routinely using the PB CD34+ cell count to initiate apheresis, 51% (22/43) first sent the test before the patient presented for collection. Although more than 90% of centers used a laboratory test to trigger apheresis in cytokine-mobilized (44/48) or chemomobilized patients (50/53), only 57% (30/53) used a laboratory trigger if the patient was mobilized with granulocyte colony-stimulating factor plus plerixafor. Forty-two percent (21/50) of programs that routinely measured the PB CD34+ count before collection and discontinued further HPC(A) collection based on product CD34+ cell yield also stopped if the PB CD34+ value before apheresis was considered too low to proceed. Most programs use the PB CD34+ cell count to trigger autologous HPC(A) collection. Some centers also use this

  1. Peripheral blood CD34+ cell count as a predictor of adequacy of hematopoietic stem cell collection for autologous transplantation

    Directory of Open Access Journals (Sweden)

    Combariza, Juan F.

    2016-10-01

    Full Text Available Introduction: In order to carry out an autologous transplantation, hematopoietic stem cells should be mobilized to peripheral blood and later collected by apheresis. The CD34+ cell count is a tool to establish the optimal time to begin the apheresis procedure. Objective: To evaluate the association between peripheral blood CD34+ cell count and the successful collection of hematopoietic stem cells. Materials and methods: A predictive test evaluation study was carried out to establish the usefulness of peripheral blood CD34+ cell count as a predictor of successful stem cell collection in patients that will receive an autologous transplantation. Results: 77 patients were included (median age: 49 years; range: 5-66. The predominant baseline diagnosis was lymphoma (53.2 %. The percentage of patients with successful harvest of hematopoietic stem cells was proportional to the number of CD34+cells in peripheral blood at the end of the mobilization procedure. We propose that more than 15 CD34+cells/μL must be present in order to achieve an adequate collection of hematopoietic stem cells. Conclusion: Peripheral blood CD34+ cell count is a useful tool to predict the successful collection of hematopoietic stem cells.

  2. Enhanced Charge Collection with Passivation Layers in Perovskite Solar Cells.

    Science.gov (United States)

    Lee, Yong Hui; Luo, Jingshan; Son, Min-Kyu; Gao, Peng; Cho, Kyung Taek; Seo, Jiyoun; Zakeeruddin, Shaik M; Grätzel, Michael; Nazeeruddin, Mohammad Khaja

    2016-05-01

    The Al2 O3 passivation layer is beneficial for mesoporous TiO2 -based perovskite solar cells when it is deposited selectively on the compact TiO2 surface. Such a passivation layer suppressing surface recombination can be formed by thermal decomposition of the perovskite layer during post-annealing.

  3. Adrenoceptors in renal medullary collecting duct (RMCD) cells

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, D.; Garg, L.C. (Univ. of Florida, Gainesville (United States))

    1990-02-26

    Recently, the authors have reported that specific, saturable and high affinity alpha{sub 1} adrenoceptors, linked to phosphoinositide messenger system, are present in the RMCD cells. In order to determine if alpha{sub 2} adrenoceptors are also present in RMCD cells, the authors measured the specific binding of ({sup 3}H)rauwolscine, an d{sub 2} adrenergic antagonist, to RMCD cells isolated from the inner medulla of the rabbit kidney. Binding of ({sup 3}H)rauwolscine to the homogenates of RMCD cells was measured in the absence (total binding) and the presence (non-specific binding) of 100 {mu}M phentolamine. The specific binding (the difference between total and non-specific binding) was measured at various concentrations of ({sup 3}H)rauwolscine. The interpolated values (fmol/mg protein) are from a curve generated using the EBDA program to analyze data from 3 animals. The apparent K{sub d} and B{sub max} of({sup 3}H)rauwolscine was 3.56 nM and 29 fmol/mg, respectively. Yohimbine inhibited binding of ({sup 3}H)rauwolscine with an IC{sub 50} of 5 {times} 10{sup {minus}9} M. Prazosin which was much less effective in displacing ({sup 3}H) rauwolscine, had a IC{sub 50} of 10{sup {minus}5} M. The authors conclude that in addition to alpha{sub 1} adrenoceptors, the specific, saturable and high affinity alpha{sub 2} adrenoceptors are also present on RMCD cells.

  4. Analysis of the inner collection efficiency in hybrid silicon solar cells

    OpenAIRE

    Nubile, P.; Torres, P; Hof, Ch.; Fischer, D.

    2008-01-01

    The collection of photogenerated carriers in hybrid silicon solar cells structures were determined by the DICE (dynamic inner collection efficiency) technique. The hybrid solar cells have a microcrystalline n-type emitter and a crystalline p-type base. Cells with amorphous buffers of several thickness and p+ back surface field microcrystalline layers were also studied. Spectral response and reflectivity were measured for each sample in order to obtain the internal spectral response or quantum...

  5. Comparison of two double red cell collection settings on Fenwal Alyx apheresis instrument.

    Science.gov (United States)

    Burgstaler, Edwin A; Duffy, Kimberly J; Gandhi, Manish J

    2017-02-09

    The Fenwal Alyx for collecting double red cell products has two red cell volume collection settings: fixed collection target of 360 ml (180 ml/unit) and a variable target of collecting either 400 or 360 ml (200 or 180 ml/unit), where the machine aims for the higher possible collection target. We retrospectively compared the two collection targets for the RBC content, donor time, technician time, and collection efficiency. We compared 18 fixed (F) target collections to 40 variable (V) target collections. All collections were performed as per the manufacturer's recommendations on Alyx and donors met the manufacturer's eligibility criteria. There was no significant difference in average whole blood processed (F: 963 ml, V: 1,000 ml); donor time (F: 43 min, V: 45 min) or technician time (F: 64 min, V: 64 min). There was a significant difference in unit volume (F: 283 ml, V: 300 ml); grams Hb/unit (F: 53 g, V: 57 g); ml RBC/unit (F: 157 ml, V: 167 ml); and RBC recovery (F: 87.8%, V: 88.9%). The fixed target had a significantly lower frequency of products with ≥51 g Hb (80.6%) than variable target (96.3%) and ≥153 ml RBC/unit (F: 55.6%, V: 96.3%). In conclusion, the variable target efficiently allows collections of products with higher red cell volume and hemoglobin without a significant increase in collection and processing time.

  6. The Cell Collective: Toward an open and collaborative approach to systems biology

    OpenAIRE

    2012-01-01

    Abstract Background Despite decades of new discoveries in biomedical research, the overwhelming complexity of cells has been a significant barrier to a fundamental understanding of how cells work as a whole. As such, the holistic study of biochemical pathways requires computer modeling. Due to the complexity of cells, it is not feasible for one person or group to model the cell in its entirety. Results The Cell Collective is a platform that allows the world-wide scientific community to create...

  7. Temporal modulation of collective cell behavior controls vascular network topology.

    Science.gov (United States)

    Kur, Esther; Kim, Jiha; Tata, Aleksandra; Comin, Cesar H; Harrington, Kyle I; Costa, Luciano da F; Bentley, Katie; Gu, Chenghua

    2016-02-24

    Vascular network density determines the amount of oxygen and nutrients delivered to host tissues, but how the vast diversity of densities is generated is unknown. Reiterations of endothelial-tip-cell selection, sprout extension and anastomosis are the basis for vascular network generation, a process governed by the VEGF/Notch feedback loop. Here, we find that temporal regulation of this feedback loop, a previously unexplored dimension, is the key mechanism to determine vascular density. Iterating between computational modeling and in vivo live imaging, we demonstrate that the rate of tip-cell selection determines the length of linear sprout extension at the expense of branching, dictating network density. We provide the first example of a host tissue-derived signal (Semaphorin3E-Plexin-D1) that accelerates tip cell selection rate, yielding a dense network. We propose that temporal regulation of this critical, iterative aspect of network formation could be a general mechanism, and additional temporal regulators may exist to sculpt vascular topology.

  8. Phase Diagram of Collective Motion of Bacterial Cells in a Shallow Circular Pool

    CERN Document Server

    Wakita, Jun-ichi; Yamamoto, Ken; Katori, Makoto; Yamada, Yasuyuki

    2015-01-01

    The collective motion of bacterial cells in a shallow circular pool is systematically studied using the bacterial species $Bacillus$ $subtilis$. The ratio of cell length to pool diameter (i.e., the reduced cell length) ranges from 0.06 to 0.43 in our experiments. Bacterial cells in a circular pool show various types of collective motion depending on the cell density in the pool and the reduced cell length. The motion is classified into six types, which we call random motion, turbulent motion, one-way rotational motion, two-way rotational motion, random oscillatory motion, and ordered oscillatory motion. Two critical values of reduced cell lengths are evaluated, at which drastic changes in collective motion are induced. A phase diagram is proposed in which the six phases are arranged.

  9. Dentinal tubules driven wetting of dentin: Cassie-Baxter modelling

    Science.gov (United States)

    Ramos, S. M. M.; Alderete, L.; Farge, P.

    2009-10-01

    We investigate the wetting properties of dentin surfaces submitted to a phosphoric acid etching followed by an air drying procedure, as in clinical situations of adhesive dentistry. The surface topography of the etched surfaces was characterized by AFM, and the wetting properties of water on these rough and heterogeneous surfaces were studied, by contact angle measurements. We showed that the contact angle increases with the acid exposure time and consequently with both surface roughness and the organic-mineral ratio of the dentin components. From the whole results, obtained on dentin and also on synthesized hydroxyapatites samples, we inferred a water contact angle of ˜ 133° on the dentinal tubule. These experimental results may be described by the Cassie-Baxter approach, and it is suggested that small air pockets could be formed inside the dentinal tubules.

  10. Effect of Tamoxifen on Seminiferous Tubules Structure during Pregnancy in Adult Mice

    Directory of Open Access Journals (Sweden)

    J Soleimani Rad

    2016-03-01

    Full Text Available Introduction: Tamoxifen is a nonsteroidal drug which mainly treats breast cancer. It is also applied for stimulation of ovulation and remedy of infertility. Regarding the tamoxifen binding to estrogen receptors and the possible role of estrogens in spermatogenesis, the present study aimed to histologically evaluate spermatogenesis in the seminiferous ducts of mice, whose mothers had received tamoxifen during pregnancy. Methods: In the present study, 30 female and 15 male mice of NMRI race were selected for mating. Since 13th day of pregnancy, the experimental group received tamoxifen with the dosage of 5 mg/kg intra-peritoneally for 7 days, wherease the control group received normal saline. After childbirth of the mated mice, male infants were selected and monitored in the standard laboratory conditions. After reaching the age of puberty (6-8Weeks, adult mice were sacrificed by the cervical dislocation, and the testes were removed for histological evaluation of spermatogenesis. After routine histological processing, the samples were studied by the light microscope. Results: Histological studies showed that spermatogenic and Sertoli cells in the seminiferous tubules in control and experimental groups were significantly different, though no difference was observed in the number of Leydig cells in the both groups. Conclusion: The findings of the present study showed that tamoxifen exposure during development can cause histological changes in the seminiferous tubules, which can lead to infertility in the male rat.

  11. Prevalence of outer retinal tubulation in eyes with choroidal neovascularization

    OpenAIRE

    Giachetti Filho, Richard Geraldo; Zacharias,Leandro Cabral; Monteiro,Thaís Vera; Preti, Rony Carlos; Pimentel, Sérgio Gianoti

    2016-01-01

    Background Outer retinal tubulations (ORTs) are branching tubular structures located in the outer nuclear layer of the retina. The goal of this study is to determine the prevalence of ORTs observed in eyes with choroidal neovascularization (CNV) undergoing treatment with anti-angiogenic intravitreous injection (IVI) with anti-VEGF (vascular endothelial growth factor) at the Ophthalmology Department of a tertiary hospital in São Paulo, Brazil. Methods This is a descriptive study based on medic...

  12. Effect of testicular capsulotomy on lipid droplets in the seminiferous tubules of rats

    Institute of Scientific and Technical Information of China (English)

    Da-Nian QIN; Mary A. Lung

    2001-01-01

    Aim: In order to reveal the histochemical alteration that might occur during the processes of the spermatogenic dis mption induced by testicular capsulotomy, the location and alteration of lipid droplets in the seminiferous tubules were observed in the present study. Methods: Osmium tetroxide was used to demonstrate the lipid droplets in the semi niferous tubules of capsulotomized and sham-operated control testes. Results: In the seminiferous tubules of the sham-operated rat testes, many small lipid droplets were located close to the basement membrane of the seminiferous tubules. But for the capsulotomized testes, the lipid droplets in the seminiferous tubules had increased in size and num ber, with many lipid droplets migrated towards the lumen of the tubules. Conclusion: The results indicated that a progressive fatty degeneration occurred in the seminiferous tubules after testicular capsulotomy.

  13. Current Collecting Grids for ITO-Free Solar Cells

    DEFF Research Database (Denmark)

    Galagan, Yulia; Zimmermann, Birger; Coenen, Erica W. C.

    2012-01-01

    enabling the identification of the most rational grid structure is presented. Both optical and light beam induced current (LBIC) mapping of the devices are used to support the power loss model and to follow the evolution of the performance over time. Current generation is found to be evenly distributed......Indium-tin-oxide (ITO) free polymer solar cells prepared by ink jet printing a composite front electrode comprising silver grid lines and a semitransparent PEDOT:PSS conductor are demonstrated. The effect of grid line density is explored for a large series of devices and a careful modeling study...

  14. Regulation of the mitochondrial permeability transition in kidney proximal tubules and its alteration during hypoxia-reoxygenation.

    Science.gov (United States)

    Feldkamp, Thorsten; Park, Jeong Soon; Pasupulati, Ratna; Amora, Daniela; Roeser, Nancy F; Venkatachalam, M A; Weinberg, Joel M

    2009-12-01

    Development of the mitochondrial permeability transition (MPT) can importantly contribute to lethal cell injury from both necrosis and apoptosis, but its role varies considerably with both the type of cell and type of injury, and it can be strongly opposed by the normally abundant endogenous metabolites ADP and Mg(2+). To better characterize the MPT in kidney proximal tubule cells and assess its contribution to injury to them, we have refined and validated approaches to follow the process in whole kidney proximal tubules and studied its regulation in normoxic tubules and after hypoxia-reoxygenation (H/R). Physiological levels of ADP and Mg(2+) greatly decreased sensitivity to the MPT. Inhibition of cyclophilin D by cyclosporine A (CsA) effectively opposed the MPT only in the presence of ADP and/or Mg(2+). Nonesterified fatty acids (NEFA) had a large role in the decreased resistance to the MPT seen after H/R irrespective of the available substrate or the presence of ADP, Mg(2+), or CsA, but removal of NEFA was less effective at restoring normal resistance to the MPT in the presence of electron transport complex I-dependent substrates than with succinate. The data indicate that the NEFA accumulation that occurs during both hypoxia in vitro and ischemic acute kidney injury in vivo is a critical sensitizing factor for the MPT that overcomes the antagonistic effect of endogenous metabolites and cyclophilin D inhibition, particularly in the presence of complex I-dependent substrates, which predominate in vivo.

  15. Dysferlin and myoferlin regulate transverse tubule formation and glycerol sensitivity.

    Science.gov (United States)

    Demonbreun, Alexis R; Rossi, Ann E; Alvarez, Manuel G; Swanson, Kaitlin E; Deveaux, H Kieran; Earley, Judy U; Hadhazy, Michele; Vohra, Ravneet; Walter, Glenn A; Pytel, Peter; McNally, Elizabeth M

    2014-01-01

    Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy.

  16. A photoactivatable nanopatterned substrate for analyzing collective cell migration with precisely tuned cell-extracellular matrix ligand interactions.

    Directory of Open Access Journals (Sweden)

    Yoshihisa Shimizu

    Full Text Available Collective cell migration is involved in many biological and pathological processes. Various factors have been shown to regulate the decision to migrate collectively or individually, but the impact of cell-extracellular matrix (ECM interactions is still debated. Here, we developed a method for analyzing collective cell migration by precisely tuning the interactions between cells and ECM ligands. Gold nanoparticles are arrayed on a glass substrate with a defined nanometer spacing by block copolymer micellar nanolithography (BCML, and photocleavable poly(ethylene glycol (Mw  =  12 kDa, PEG12K and a cyclic RGD peptide, as an ECM ligand, are immobilized on this substrate. The remaining glass regions are passivated with PEG2K-silane to make cells interact with the surface via the nanoperiodically presented cyclic RGD ligands upon the photocleavage of PEG12K. On this nanostructured substrate, HeLa cells are first patterned in photo-illuminated regions, and cell migration is induced by a second photocleavage of the surrounding PEG12K. The HeLa cells gradually lose their cell-cell contacts and become disconnected on the nanopatterned substrate with 10-nm particles and 57-nm spacing, in contrast to their behavior on the homogenous substrate. Interestingly, the relationship between the observed migration collectivity and the cell-ECM ligand interactions is the opposite of that expected based on conventional soft matter models. It is likely that the reduced phosphorylation at tyrosine-861 of focal adhesion kinase (FAK on the nanopatterned surface is responsible for this unique migration behavior. These results demonstrate the usefulness of the presented method in understanding the process of determining collective and non-collective migration features in defined micro- and nano-environments and resolving the crosstalk between cell-cell and cell-ECM adhesions.

  17. Podocyturia parallels proximal tubule dysfunction in type 2 diabetes mellitus patients independently of albuminuria and renal function decline: A cross-sectional study.

    Science.gov (United States)

    Petrica, Ligia; Vlad, Mihaela; Vlad, Adrian; Gluhovschi, Gheorghe; Gadalean, Florica; Dumitrascu, Victor; Popescu, Roxana; Gluhovschi, Cristina; Matusz, Petru; Velciov, Silvia; Bob, Flaviu; Ursoniu, Sorin; Vlad, Daliborca

    2017-09-01

    Detection of podocytes in the urine of patients with type 2 diabetes may indicate severe injury to the podocytes. In the course of type 2 diabetes the proximal tubule is involved in urinary albumin processing. We studied the significance of podocyturia in relation with proximal tubule dysfunction in type 2 diabetes. A total of 86 patients with type 2 diabetes (34-normoalbuminuria; 30-microalbuminuria; 22-macroalbuminuria) and 28 healthy subjects were enrolled in the study and assessed concerning urinary podocytes, podocyte-associated molecules, and biomarkers of proximal tubule dysfunction. Urinary podocytes were examined in cell cultures by utilizing monoclonal antibodies against podocalyxin and synaptopodin. Podocytes were detected in the urine of 10% of the healthy controls, 24% of the normoalbuminuric, 40% of the microalbuminuric, and 82% of the macroalbuminuric patients. In multivariate logistic regression analysis, urinary podocytes correlated with urinary albumin:creatinine ratio (p=0.006), urinary nephrin/creat (p=0.001), urinary vascular endothelial growth factor/creat (p=0.001), urinary kidney injury molecule-1/creat (p=0.003), cystatin C (p=0.001), urinary advanced glycation end-products (p=0.002), eGFR (p=0.001). In patients with type 2 diabetes podocyturia parallels proximal tubule dysfunction independently of albuminuria and renal function decline. Advanced glycation end-products may impact the podocytes and the proximal tubule. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Current collecting grids for ITO-free solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Galagan, Yulia; Gorter, Harrie; Sabik, Sami; Andriessen, Ronn [Holst Centre, High Tech Campus 31, Eindhoven (Netherlands); Zimmermann, Birger [Fraunhofer Institute for Solar Energy Systems ISE, Freiburg (Germany); Coenen, Erica W.C. [TNO Technical Sciences, Eindhoven (Netherlands); Joergensen, Mikkel; Krebs, Frederik C. [Risoe National Laboratory for Sustainable Energy, Technical University of Denmark, Roskilde (Denmark); Tanenbaum, David M. [Risoe National Laboratory for Sustainable Energy, Technical University of Denmark, Roskilde (Denmark); Department of Physics and Astronomy, Pomona College, Claremont, CA (United States); Slooff, Lenneke H.; Veenstra, Sjoerd C.; Kroon, Jan M. [Energy Research Centre of the Netherlands (ECN), Petten (Netherlands)

    2012-01-15

    Indium-tin-oxide (ITO) free polymer solar cells prepared by ink jet printing a composite front electrode comprising silver grid lines and a semitransparent PEDOT:PSS conductor are demonstrated. The effect of grid line density is explored for a large series of devices and a careful modeling study enabling the identification of the most rational grid structure is presented. Both optical and light beam induced current (LBIC) mapping of the devices are used to support the power loss model and to follow the evolution of the performance over time. Current generation is found to be evenly distributed over the active area initially progressing to a larger graduation in areas with different performance. Over time coating defects also become much more apparent in the LBIC images. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  19. Analysis of CD34+ cell collection using two mobilization regimens for newly diagnosed multiple myeloma patients reveals the separate impact of mobilization and collection variables.

    Science.gov (United States)

    Abuabdou, Ahmed; Rosenbaum, Eric R; Usmani, Saad Zafar; Barlogie, Bart; Cottler-Fox, Michele

    2014-10-01

    Mobilization regimens for CD34+ cells have generally been judged successful based on the number of cells collected without evaluating mobilization separately from collection. Using retrospective data for patients who collected CD34+ cells on Total Therapy protocols 3a/3b (VTD-PACE) and Total Therapy 4/5 using a novel regimen that added low dose melphalan to VTD-PACE (MVTD-PACE), we analyzed mobilization and collection variables separately. A significant difference favoring MVDT-PACE was found in mean CD34+ cells/µL on day 2 of collection and in mean ratio of CD34+ cells/µL on day 2 to day 1. However, because apheresis variables and growth factor dose during collection were manipulated to optimize individual collections, the two regimens were not significantly different when the mean total CD34+ cells ×10(6) /kg collected was compared. Thus, when evaluating a chemotherapy regimen or new growth factor for mobilization, it is important to realize that total CD34+ cells collected is dependent on both mobilization and collection variables.

  20. Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin beta 1 and PI3K

    OpenAIRE

    YAMAGUCHI, Naoya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2015-01-01

    Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that f...

  1. Activated extracellular signal-regulated kinases are necessary and sufficient to initiate tubulogenesis in renal tubular MDCK strain I cell cysts.

    Science.gov (United States)

    Hellman, Nathan E; Greco, Andres J; Rogers, Katherine K; Kanchagar, Chitra; Balkovetz, Daniel F; Lipschutz, Joshua H

    2005-10-01

    A classic in vitro model of renal cyst and tubule formation utilizes the Madin-Darby canine kidney (MDCK) cell line, of which two strains exist. Most cyst and tubule formation studies that utilized MDCK cells have been performed with MDCK strain II cells. MDCK strain II cells form hollow cysts in a three-dimensional collagen matrix over 10 days and tubulate in response to hepatocyte growth factor, which increases levels of active (phosphorylated) ERK1/2. In this study, we demonstrate that MDCK strain I cells also form cysts when grown in a collagen matrix; however, MDCK strain I cell cysts spontaneously initiate the primary steps in tubulogenesis. Analysis of time-lapse microscopy of both MDCK strain I and strain II cell cysts during the initial stages of tubulogenesis demonstrates a highly dynamic process with cellular extensions and retractions occurring rapidly and continuously. MDCK strain I cell cysts can spontaneously initiate tubulogenesis mainly because of relatively higher levels of active ERK in MDCK strain I, compared with strain II, cells. The presence of either of two distinct inhibitors of ERK activation (UO126 and PD09059) prevents tubulogenesis from occurring spontaneously in MDCK strain I cell cysts and, in response to hepatocyte growth factor, in strain II cell cysts. The difference between MDCK strain I and strain II cell lines is likely explained by differing embryological origins, with strain I cells being of collecting duct, and hence ureteric bud, origin. Ureteric bud cells also have high levels of active ERK and spontaneously tubulate in our in vitro collagen gel system, with tubulogenesis inhibited by UO126 and PD09059. These results suggest that a seminal event in kidney development may be the activation of ERK in the mesonephric duct/ureteric bud cells destined to form the collecting tubules.

  2. The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Gibbs

    2014-01-01

    Full Text Available DNM2 is a ubiquitously expressed GTPase that regulates multiple subcellular processes. Mutations in DNM2 are a common cause of centronuclear myopathy, a severe disorder characterized by altered skeletal muscle structure and function. The precise mechanisms underlying disease-associated DNM2 mutations are unresolved. We examined the common DNM2-S619L mutation using both in vitro and in vivo approaches. Expression of DNM2-S619L in zebrafish led to the accumulation of aberrant vesicular structures and to defective excitation-contraction coupling. Expression of DNM2-S619L in COS7 cells resulted in defective BIN1-dependent tubule formation. These data suggest that DNM2-S619L causes disease, in part, by interfering with membrane tubulation.

  3. Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis

    Science.gov (United States)

    Mei, Shuqin; Li, Lin; Wei, Qingqing; Hao, Jielu; Su, Yunchao; Mei, Changlin; Dong, Zheng

    2017-01-01

    Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases. PMID:28317867

  4. Diabetes increases facilitative glucose uptake and GLUT2 expression at the rat proximal tubule brush border membrane.

    Science.gov (United States)

    Marks, Joanne; Carvou, Nicolas J C; Debnam, Edward S; Srai, Surjit K; Unwin, Robert J

    2003-11-15

    The mechanism of renal glucose transport involves the reabsorption of filtered glucose from the proximal tubule lumen across the brush border membrane (BBM) via a sodium-dependent transporter, SGLT, and exit across the basolateral membrane via facilitative, GLUT-mediated, transport. The aim of the present study was to determine the effect of streptozotocin-induced diabetes on BBM glucose transport. We found that diabetes increased facilitative glucose transport at the BBM by 67.5 % (P < 0.05)--an effect that was abolished by overnight fasting. Western blotting and immunohistochemistry demonstrated GLUT2 expression at the BBM during diabetes, but the protein was undetectable at the BBM of control animals or diabetic animals that had been fasted overnight. Our findings indicate that streptozotocin-induced diabetes causes the insertion of GLUT2 into the BBM and this may provide a low affinity/high capacity route of entry into proximal tubule cells during hyperglycaemia.

  5. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.

    Science.gov (United States)

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation.

  6. Collecting duct-derived cells display mesenchymal stem cell properties and retain selective in vitro and in vivo epithelial capacity.

    Science.gov (United States)

    Li, Joan; Ariunbold, Usukhbayar; Suhaimi, Norseha; Sunn, Nana; Guo, Jinjin; McMahon, Jill A; McMahon, Andrew P; Little, Melissa

    2015-01-01

    We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC-like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC-like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC-like cells selectively integrated into the aquaporin 2-positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC-like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC-like cells, we further examined Hoxb7(+) fractions within the kidney across postnatal development, identifying a neonatal interstitial GFP(lo) (Hoxb7(lo)) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4(GCE/+):R26(tdTomato/+) mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair.

  7. Local pH domains regulate NHE3-mediated Na⁺ reabsorption in the renal proximal tubule.

    Science.gov (United States)

    Brasen, Jens Christian; Burford, James L; McDonough, Alicia A; Holstein-Rathlou, Niels-Henrik; Peti-Peterdi, Janos

    2014-12-01

    The proximal tubule Na(+)/H(+) exchanger 3 (NHE3), located in the apical dense microvilli (brush border), plays a major role in the reabsorption of NaCl and water in the renal proximal tubule. In response to a rise in blood pressure NHE3 redistributes in the plane of the plasma membrane to the base of the brush border, where NHE3 activity is reduced. This NHE3 redistribution is assumed to provoke pressure natriuresis; however, it is unclear how NHE3 redistribution per se reduces NHE3 activity. To investigate if the distribution of NHE3 in the brush border can change the reabsorption rate, we constructed a spatiotemporal mathematical model of NHE3-mediated Na(+) reabsorption across a proximal tubule cell and compared the model results with in vivo experiments in rats. The model predicts that when NHE3 is localized exclusively at the base of the brush border, it creates local pH microdomains that reduce NHE3 activity by >30%. We tested the model's prediction experimentally: the rat kidney cortex was loaded with the pH-sensitive fluorescent dye BCECF, and cells of the proximal tubule were imaged in vivo using confocal fluorescence microscopy before and after an increase of blood pressure by ∼50 mmHg. The experimental results supported the model by demonstrating that a rise of blood pressure induces the development of pH microdomains near the bottom of the brush border. These local changes in pH reduce NHE3 activity, which may explain the pressure natriuresis response to NHE3 redistribution.

  8. Collective motion of cells mediates segregation and pattern formation in co-cultures.

    Directory of Open Access Journals (Sweden)

    Elod Méhes

    Full Text Available Pattern formation by segregation of cell types is an important process during embryonic development. We show that an experimentally yet unexplored mechanism based on collective motility of segregating cells enhances the effects of known pattern formation mechanisms such as differential adhesion, mechanochemical interactions or cell migration directed by morphogens. To study in vitro cell segregation we use time-lapse videomicroscopy and quantitative analysis of the main features of the motion of individual cells or groups. Our observations have been extensive, typically involving the investigation of the development of patterns containing up to 200,000 cells. By either comparing keratocyte types with different collective motility characteristics or increasing cells' directional persistence by the inhibition of Rac1 GTP-ase we demonstrate that enhanced collective cell motility results in faster cell segregation leading to the formation of more extensive patterns. The growth of the characteristic scale of patterns generally follows an algebraic scaling law with exponent values up to 0.74 in the presence of collective motion, compared to significantly smaller exponents in case of diffusive motion.

  9. Light trapping in a 30-nm organic photovoltaic cell for efficient carrier collection and light absorption

    CERN Document Server

    Tsai, Cheng-Chia; Banerjee, Ashish; Osgood, Richard M; Englund, Dirk

    2012-01-01

    We describe surface patterning strategies that permit high photon-collection efficiency together with high carrier-collection efficiency in an ultra-thin planar heterojunction organic photovoltaic cell. Optimized designs reach up to 50% photon collection efficiency in a P3HT layer of only 30 nm, representing a 3- to 5-fold improvement over an unpatterned cell of the same thickness. We compare the enhancement of light confinement in the active layer with an ITO top layer for TE and TM polarized light, and demonstrate that the light absorption can increase by a factor of 2 due to a gap-plasmon mode in the active layer.

  10. In vitro analysis of cell salvage blood collection with a laparoscopic suction device.

    Science.gov (United States)

    Nagarsheth, Nimesh P; Fenske, Suzanne Silverman; Shah, Apurva; Moshier, Erin; Stahl, Rosalyn; Shander, Aryeh

    2013-01-01

    To determine whether cell salvage blood collection with a laparoscopic suction device is inferior to use of a traditional Yankauer suction device. Prospective, in vitro study. Academic teaching hospital. Individual units of donated packed red blood cells were diluted with normal saline solution to a hematocrit level of 21%. The blood was divided into 2 equal parts and then suctioned with either a laparoscopic suction device or a Yankauer plastic suction catheter tip connected to double-lumen cell salvage tubing with a diluted heparin drip and a vacuum pressure of 100 mm Hg. Collected blood was processed with a cell salvage device. Red blood cell volume was calculated by multiplying the hematocrit level by the total volume of blood product at the time of testing. Mean hemolysis indexes were compared between the laparoscopic and Yankauer method of blood collection by use of a 2-sample t test. Assuming a clinically acceptable limit of loss to be 7%, percent loss in red blood cell volume was tested with a 95% one-sided confidence limit to assess noninferiority. The mean hemolysis index was 43.33 with laparoscopic suction method and 34.67 with the Yankauer suction method. The mean difference was 8.67 and was not considered significant (p = .074). The percent loss in red blood cell volume after collection and cell salvage processing was 33.2% with the laparoscopic suction method and 29.57% with the Yankauer method. The mean difference was 3.63% and was within the acceptable 7% loss limit for noninferiority (p = .0278). Laparoscopic blood collection is not inferior to the standard Yankauer method for cell salvage collection. Published by Elsevier Inc.

  11. Effect of dentinal tubules and resin-based endodontic sealers on fracture properties of root dentin.

    Science.gov (United States)

    Jainaen, Angsana; Palamara, Joseph E A; Messer, Harold H

    2009-10-01

    To investigate the role of dentinal tubules in the fracture properties of human root dentin and whether resin-filled dentinal tubules can enhance fracture resistance. Crack propagation in human root dentin was investigated in 200 microm thick longitudinal samples and examined by light and scanning electron microscopy. 30 maxillary premolar teeth were prepared for work of fracture (Wf) test at different tubule orientations, one perpendicular and two parallel to dentinal tubules. Another 40 single canal premolars were randomly divided into four groups of 10 each: intact dentin, prepared but unobturated canal, canal obturated with epoxy rein (AH Plus/gutta percha), or with UDMA resin sealer (Resilon/RealSeal. The samples were prepared for Wf test parallel to dentinal tubules. Wf was compared under ANOVA with statistical significance set at pcanal preparation nor obturation using epoxy- or UDMA-based resins as sealer cements substantially influenced fracture properties of root dentin, despite extensive infiltration of dentinal tubules by both sealer cements.

  12. ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells.

    Science.gov (United States)

    Ding, You-Quan; Li, Xuan-Yang; Xia, Guan-Nan; Ren, Hong-Yi; Zhou, Xin-Fu; Su, Bing-Yin; Qi, Jian-Guo

    2016-10-01

    Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since "the yin and yang of neurotrophin action" has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of "the yin and yang of neurotrophin action" and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.

  13. Fate of iPSCs Derived from Azoospermic and Fertile Men following Xenotransplantation to Murine Seminiferous Tubules

    Directory of Open Access Journals (Sweden)

    Cyril Ramathal

    2014-05-01

    Full Text Available Historically, spontaneous deletions and insertions have provided means to probe germline developmental genetics in Drosophila, mouse and other species. Here, induced pluripotent stem cell (iPSC lines were derived from infertile men with deletions that encompass three Y chromosome azoospermia factor (AZF regions and are associated with production of few or no sperm but normal somatic development. AZF-deleted iPSC lines were compromised in germ cell development in vitro. Undifferentiated iPSCs transplanted directly into murine seminiferous tubules differentiated extensively to germ-cell-like cells (GCLCs that localized near the basement membrane, demonstrated morphology indistinguishable from fetal germ cells, and expressed germ-cell-specific proteins diagnostic of primordial germ cells. Alternatively, all iPSCs that exited tubules formed primitive tumors. iPSCs with AZF deletions produced significantly fewer GCLCs in vivo with distinct defects in gene expression. Findings indicate that xenotransplantation of human iPSCs directs germ cell differentiation in a manner dependent on donor genetic status.

  14. Proteomic profiling and pathway analysis of the response of rat renal proximal convoluted tubules to metabolic acidosis.

    Science.gov (United States)

    Schauer, Kevin L; Freund, Dana M; Prenni, Jessica E; Curthoys, Norman P

    2013-09-01

    Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis.

  15. Proteomic profiling and pathway analysis of the response of rat renal proximal convoluted tubules to metabolic acidosis

    Science.gov (United States)

    Schauer, Kevin L.; Freund, Dana M.; Prenni, Jessica E.

    2013-01-01

    Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis. PMID:23804448

  16. Rhodnius prolixus Malpighian tubules and control of diuresis by neurohormones

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2007-03-01

    Full Text Available Rhodnius prolixus Malpighian tubules (MTs are a good model for fluid and ion secretion studies in view of the dramatic postprandial diuresis, which follows its massive blood meals. Ingestion of a blood meal equals to 10-12 times their initial body mass, leads to rapid activation of high output by excretory system, which eliminates 40-50% of the fluid mass. Secretion of ions and water is stimulated 1000-fold by serotonin and diuretic hormone. These hormones cooperate synergistically to activate adenylate cyclase activity from MTs cells, which increase the level of intracellular cAMP. The anti-diuretic hormones have also an important role in the fluid maintenance of Rhodnius prolixus. Several hours after insect feeding occurs a reduction in urine flow, that has been thought to result from a decreased diuretic hormone release or from a novel mechanism of anti-diuresis involving insect cardioacceleratory peptide 2b (CAP2b and cyclic GMP. In this article it is discussed how the hormone regulation of fluid transport is done in Rhodnius prolixus MTs.Os túbulos de Malpighi (TMs de Rhodnius prolixus são reconhecidos por serem excelentes modelos para o estudo da secreção de fluidos e íons devido a grande diurese que ocorre quando esses animais se alimentam de sangue. O inseto, após alimentação, pode aumentar seu peso corporal inicial em até 10-12 vezes, o que leva a rápida ativação do sistema excretor, que elimina 40-50% do fluido corporal. A secreção de íons e água é estimulada 1000 vezes pela serotonina e pelos hormônios diuréticos. Esses hormônios agem sinergicamente ativando a adenil ciclase das células dos TMs, aumentando os níveis intracelulares de AMPc. Os hormônios anti-diuréticos também têm um importante papel na manutenção dos fluídos corporais do Rhodnius prolixus. Várias horas após a alimentação do inseto ocorre uma redução do fluxo urinário, o que foi sugerido ser decorrente da diminuição da libera

  17. Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.

    Science.gov (United States)

    Fugier, Charlotte; Klein, Arnaud F; Hammer, Caroline; Vassilopoulos, Stéphane; Ivarsson, Ylva; Toussaint, Anne; Tosch, Valérie; Vignaud, Alban; Ferry, Arnaud; Messaddeq, Nadia; Kokunai, Yosuke; Tsuburaya, Rie; de la Grange, Pierre; Dembele, Doulaye; Francois, Virginie; Precigout, Guillaume; Boulade-Ladame, Charlotte; Hummel, Marie-Christine; Lopez de Munain, Adolfo; Sergeant, Nicolas; Laquerrière, Annie; Thibault, Christelle; Deryckere, François; Auboeuf, Didier; Garcia, Luis; Zimmermann, Pascale; Udd, Bjarne; Schoser, Benedikt; Takahashi, Masanori P; Nishino, Ichizo; Bassez, Guillaume; Laporte, Jocelyn; Furling, Denis; Charlet-Berguerand, Nicolas

    2011-06-01

    Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

  18. Changes in cellular composition of kidney collecting duct cells in rats with lithium-induced NDI.

    Science.gov (United States)

    Christensen, Birgitte Mønster; Marples, David; Kim, Young-Hee; Wang, Weidong; Frøkiaer, Jørgen; Nielsen, Søren

    2004-04-01

    Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H(+)]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H(+)]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 +/- 3.4 vs. 62 +/- 1.8% of controls (P diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus.

  19. Cellular regulation of follicle-stimulating hormone (FSH) binding in rat seminiferous tubules

    Energy Technology Data Exchange (ETDEWEB)

    Kangasniemi, M.; Kaipia, A.; Toppari, J.; Perheentupa, A.; Huhtaniemi, I.; Parvinen, M. (Univ. of Turku (Finland))

    1990-07-01

    Stage-specific binding of follicle-stimulating hormone (FSH) was measured in rat seminiferous tubules. The binding in single-point assays was over 3-fold higher (P less than 0.05) in stages XIII to I than in stages VI to VII of the epithelial cycle. No difference was found between the equilibrium association constants (Ka) of FSH binding in stages XIV to IV (10 +/- 1.9 X 10(9) 1/mol) and VII to VIII (9.2 +/- 0.6 X 10(9) 1/mol, mean +/- SEM, n = 5). In another experiment, the testes were dosed locally with 3 Gy of 4 MV x-irradiation to selectively lower the number of spermatogonia. After irradiation, FSH binding in staged seminiferous tubule segments was measured when the desired types of spermatogenic cells were reduced in number. Seven days after irradiation when differentiating spermatogonia and preleptotene spermatocytes were reduced in number, FSH binding was decreased in all stages of the cycle, but the cyclic variation remained. Seventeen days after irradiation when intermediate and type B spermatogonia and spermatocytes up to diplotene of stage XIII showed low numbers, FSH binding was decreased in all stages of the cycle and the stage-dependent variation disappeared. At 38 days when pachytene spermatocytes and early spermatids were reduced in number, similar results were found. But at 52 days postirradiation when all spermatids were low in number, FSH binding was slightly elevated compared with days 17 and 38. There were no significant differences in serum FSH or LH levels between irradiated and non-irradiated animals. These findings suggest that all spermatogenic cell types may stimulate FSH binding in the Sertoli cells.

  20. Preliminary Observation on the Influence of Tumor Osseous Metastasis on Autologous Peripheral Blood Stem Cell Collection

    Institute of Scientific and Technical Information of China (English)

    Xiaoming Si; Wenchao Liu; Yan Xue; Hongmei Zhang; Rong Sheng; Ying Huang; Jie Cheng

    2007-01-01

    OBJECTIVE To examine the influence of tumor osseous metastasis on the patients undergoing autologous peripheral blood stem cell collection. METHODS A total of 36 patients with malignant diseases who received an autologous peripheral blood stem cell transplantation, during a period from April 2004 to June 2006, were chosen. The patients were divided into two groups, I.e. Group A were patients with a complication of tumor osseous metastasis, and group B were without metastasis. Both groups were treated with Taxotere 120 mg/m2 plus granulocyte colony-stimulating factor (G-CSF) 5 ug/kg/d, for a mobilization regimen. A blood cell separator was used to collect the mononuclear cells. The proportion of harvested CD34+ cells in the peripheral blood and the collected mononuclear cells were detected by flow cytometry. The number of CD34+ cells was used to determine the difference in the nature of the collections between the two groups. RESULTS After mobilization in groups A and B, the number of the peripheral blood mononuclear cells (PBMC) was 39.3 ±14.7% and 41.±12.4 % and the proportion of CD34 + cells was 0.16±0.07% and 0.17 ± 0.10%, respectively. Following administration of the drugs, there was no significant difference between the number of harvested PBMC and CD34+ cells of the two groups, I.e., 3.47 ± 1.16 x 108/Kg and 2.52 ± 1.43 × 106/Kg in group A and 4.02 ± 1.31 × 108/Kg and 2.73 ± 1.87 x 108/Kg in group B, respectively. CONCLUSION Osseous metastasis, as a single factor, may have no impact on mobilization and harvesting of hematopoietic stem cells and their engraftment after autotransplantation.

  1. The effect of exercise training on transverse tubules in normal, remodeled, and reverse remodeled hearts.

    Science.gov (United States)

    Kemi, Ole J; Hoydal, Morten A; Macquaide, Niall; Haram, Per M; Koch, Lauren G; Britton, Steven L; Ellingsen, Oyvind; Smith, Godfrey L; Wisloff, Ulrik

    2011-09-01

    The response of transverse (T)-tubules to exercise training in health and disease remains unclear. Therefore, we studied the effect of exercise training on the density and spacing of left ventricle cardiomyocyte T-tubules in normal and remodeled hearts that associate with detubulation, by confocal laser scanning microscopy. First, exercise training in normal rats increased cardiomyocyte volume by 16% (P hypertrophy. Next, we studied T-tubules in a rat model of metabolic syndrome with pressure overload-induced concentric left ventricle hypertrophy, evidenced by 15% (P Exercise training further increased cardiomyocyte volume by 8% (P eccentric and concentric hypertrophy and 55% (P Exercise training reversed 50% (P hypertrophy, whereas the T-tubule density increased by 40% (P hypertrophy associated with conserved T-tubule spacing (~1.8-1.9 µm), whereas in pathologic hypertrophy, T-tubules appeared disorganized without regular spacing. In conclusion, cardiomyocytes maintain the relative T-tubule density during physiologic hypertrophy and after mild concentric pathologic hypertrophy, whereas after severe pathologic remodeling with a substantial loss of T-tubules; exercise training reverses the remodeling and partly corrects the T-tubule density.

  2. Number of malpighian tubules in larvae and adults of stingless bees (Hymenoptera: Apidae) from Amazonia.

    Science.gov (United States)

    Barbosa-Costa, K; Kerr, W E; Carvalho-Zilse, G A

    2012-02-01

    The number of Malpighian tubules in larvae and adults of bees is variable. Larvae of Apis mellifera L. have four Malpighian tubules, while adults have 100 tubules. In stingless bees, this number varies from four to eight. The objectives of this study were to provide characteristics of the Malpighian tubules as well as to quantify their number in larvae and adults of six species of Meliponinae, Melipona seminigra merrillae Cockerell, Melipona compressipes manaosensis Schwarz, Melipona rufiventris Lepeletier, Scaptotrigona Moure, Frieseomelitta Ihering, and Trigona williana Friese. Malpighian tubules were dissected from larvae and adults, measured, quantified, and maintained in microtubes with Dietrich's solution. The numbers of Malpighian tubules were constant only for larvae of M. rufiventris (four and eight) and Scaptotrigona sp. (four). The most frequent number of tubules in the Melipona group was seven and eight in larvae, and 70 and 90 in adults. In the Trigona group were four and 20 to 40, for larvae and adults, respectively. The results showed differences in the number of Malpighian tubules among the species analyzed and also between the larvae and adults of the same species. Despite the variation observed, species of the group Melipona always have a larger number and longer Malpighian tubules in both larvae and adults as compared to the Trigona group, which may indicate an evolutionary trend of differentiation between these groups.

  3. A dynamic paracellular pathway serves diuresis in mosquito Malpighian tubules.

    Science.gov (United States)

    Beyenbach, Klaus W

    2012-07-01

    Female mosquitoes gorge on vertebrate blood, a rich nutrient source for developing eggs, but gorging meals increase the risk of predation. Mosquitoes are quick to reduce the flight payload with a potent diuresis. Diuretic peptides of the insect kinin family induce a tenfold reduction in the paracellular resistance of Malpighian tubules and increase the paracellular permeation of Cl(-), the counterion of the transepithelial secretion of Na(+) and K(+). As a result, the transepithelial secretion of NaCl and KCl and water increases. Insect kinins signal the opening of the paracellular pathway via G protein-coupled receptors and the elevation of intracellular [Ca(2+)], which leads to the reorganization of the cytoskeleton associated with the septate junction (SJ). The reorganization may affect the septate junctional proteins that control the barrier and permselectivity properties of the paracellular pathway. The proteins involved in the embryonic formation of the SJ and in epithelial polarization are largely known for ectodermal epithelia, but the proteins that form and mediate the dynamic functions of the SJ in Malpighian tubules remain to be determined.

  4. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis.

    Science.gov (United States)

    Supatto, Willy; McMahon, Amy; Fraser, Scott E; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

  5. Principles of bone marrow processing and progenitor cell/mononuclear cell concentrate collection in a continuous flow blood cell separation system.

    Science.gov (United States)

    Hester, J P; Rondón, G; Huh, Y O; Lauppe, M J; Champlin, R E; Deisseroth, A B

    1995-08-01

    The application of continuous flow apheresis technology to processing bone marrow for collection of the mononuclear progenitor cell population appears to follow the same principles as collection of mononuclear cells from peripheral blood. Unlike peripheral blood, however, where mobilization of cells from extravascular sites during the procedures contributes significantly to the final cell yield, the entire quantity of progenitor cells available for recovery from marrow is present in the original marrow when it is pooled. The process then becomes one of attempting optimal recovery of the cells of interest while excluding contaminating erythrocytes and cells of the myeloid series. This study reports the development of a protocol for recovery of MNC, CD33+, CD34+, and CD34+/DR- cells from harvested marrow for autologous and allogeneic transplants using a continuous flow blood cell separator, the variables influencing the recovery of the cells of interest and the clinical response to infusion of the processed cells.

  6. Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

    Energy Technology Data Exchange (ETDEWEB)

    Backues, Steven K. [Department of Biochemistry, University of Wisconsin - Madison, 433 Babcock Dr., Madison, WI 53706 (United States); Bednarek, Sebastian Y., E-mail: sybednar@wisc.edu [Department of Biochemistry, University of Wisconsin - Madison, 433 Babcock Dr., Madison, WI 53706 (United States)

    2010-03-19

    The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

  7. Current blocking and current collection in CIGSe solar cells depending on sodium content

    Energy Technology Data Exchange (ETDEWEB)

    Puttnins, Stefan; Daume, Felix [Solarion AG, Leipzig (Germany); Institut fuer Experimentelle Physik II, Universitaet Leipzig (Germany); Zachmann, Hendrik; Rahm, Andreas [Solarion AG, Leipzig (Germany); Grundmann, Marius [Institut fuer Experimentelle Physik II, Universitaet Leipzig (Germany)

    2010-07-01

    IV-curves of thin film solar cells often show non-idealites like voltage dependent carrier collection and current blocking behaviour. Sodium is long known to improve the efficiency of Cu(In,Ga)Se{sub 2} solar cells by increasing V{sub OC} and FF. However, the way in which sodium influences the electrical properties is still under discussion. We investigated the influence of sodium on voltage dependent carrier collection and current blocking behaviour. Losses caused by incomplete photocurrent collection can be reduced by increased sodium content in the CIGSe layer. Current blocking behaviour like the rollover effect is less pronounced with increased sodium content. The influences were analyzed both in detailed illumination intensity and temperature dependent IV-measurements as well as by extensive statistical analysis over thousands of produced flexible CIGSe solar cells. Theoretical models for this dependency were simulated with SCAPS-1D and show good agreement with respective measurements.

  8. Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

    OpenAIRE

    Schenk, Laura K.; Bolger, Steven J.; Luginbuhl, Kelli; Gonzales, Patricia A.; Rinschen, Markus M.; Yu, Ming-Jiun; Hoffert, Jason D.; Pisitkun, Trairak; Knepper, Mark A.

    2012-01-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nucl...

  9. Loss of myoferlin redirects breast cancer cell motility towards collective migration.

    Directory of Open Access Journals (Sweden)

    Leonithas I Volakis

    Full Text Available Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET and reduced invasion through extracellular matrix (ECM. However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231(MYOF-KD leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA-231(LTVC and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA-231(MYOF-KD cells migrated directionally and collectively, while MDA-231(LTVC cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231(MYOF-KD cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA-231(MYOF-KD cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA-231(MYOF-KD tumors were highly circularized and did not invade locally into the adventia in contrast to MDA-231(LTVC-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate

  10. Emergence and Persistence of Collective Cell Migration on Small Circular Micropatterns

    CERN Document Server

    Segerer, Felix J; Alberola, Alicia Piera; Frey, Erwin; Rädler, Joachim O

    2015-01-01

    The spontaneous formation of vortices is a hallmark of collective cellular activity. Here, we study the onset and persistence of coherent angular motion (CAMo) as a function of the number of cells $N$ confined in circular micropatterns. We find that the persistence of CAMo increases with $N$ but exhibits a pronounced discontinuity accompanied by a geometric rearrangement of cells to a configuration containing a central cell. Computer simulations based on a generalized Potts model reproduce the emergence of vortex states and show in agreement with experiment that their stability depends on the interplay of spatial arrangement and internal polarization of neighboring cells. Hence, the distinct migrational states in finite size

  11. Elevated Na(+)/H(+) exchanger-1 expression enhances the metastatic collective migration of head and neck squamous cell carcinoma cells.

    Science.gov (United States)

    Kaminota, Teppei; Yano, Hajime; Shiota, Kohei; Nomura, Noriko; Yaguchi, Haruna; Kirino, Yui; Ohara, Kentaro; Tetsumura, Issei; Sanada, Tomoyoshi; Ugumori, Toru; Tanaka, Junya; Hato, Naohito

    2017-04-22

    Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na(+)/H(+) exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Inhibition of oxidative stress in high glucose-induced proximal tubule epithelial cells by breviscapine%灯盏花素抑制高糖诱导大鼠近端小管上皮细胞的氧化应激

    Institute of Scientific and Technical Information of China (English)

    杜飞; 高原; 姚君; 王兴红

    2011-01-01

    目的 探讨灯盏花素对高糖诱导的原代大鼠近端小管上皮细胞(proximal tubule epithelial cells,PTEC)氧化应激的影响.方法 原代培养大鼠PTEC并将其分为:正常对照组、高糖组、小剂量组、中剂量组、大剂量组,每组设6个复孔(n=6),作用72 h.测定PTEC培养液一氧化氮(nitrogen monoxidum,NO)、过氧化氢(hydrogen peroxide,H2O2)、过氧化氢酶(catalase,CAT)和还原型谷胱甘肽(reduced glutathione hormone,GSH)水平.结果 高糖环境时PTEC NO水平明显升高(P<0.05),小剂量组明显降低(P<0.05),中剂量和大剂量组显著性降低(P<0.01),高糖组PTEC H2O2水平显著升高(P<0.01),小剂量明显升高(P<0.05),中剂量组、大剂量组明显降低(P<0.05);高糖组PTEC CAT显著低于NC组(P<0.01),小剂量组仍明显低于高糖组(P<0.05),大剂量组显著性高于高糖组(P<0.01);高糖组PTEC GSH水平明显低于NC组(P<0.05),小剂量组仍明显较NC组低(P<0.05),中剂量组明显升高(P<0.05),大剂量显著性升高(P<0.01);结论 灯盏花素对高糖诱导的原代PTEC的氧化应激增强有明显的抑制作用;灯盏花素对DN的防治作用可能部分通过抑制PTEC的氧化应激实现的.%Objective  The present study was designed to explore the effect of breviscapine on high glucose-induced oxidative stress in primary proximal tubule epithelial cells(PTEC ) .Methods  The PTEC were primary cultured .They were randomly divided into five groups :normal controlgroup ;high glucose group ;low dose group ;middle dose group ;high dose group .All of them were cultured for 72 hours .They chemi-chromatometry were employed to assayed the levels of nitrogen monoxidum (NO ) ,hydrogen peroxide (H2O2 ) ,catalase(CAT ) and reduced glutathione hormone(GSH ) in PTEC .Results  The level of NO was significantly increased in high glucose group of PTEC ( P < 0 .05) .significantly decreased in low dose group ( P < 0 .05) and in middle

  13. Thyroid hormone modulates ClC-2 chloride channel gene expression in rat renal proximal tubules.

    Science.gov (United States)

    Santos Ornellas, D; Grozovsky, R; Goldenberg, R C; Carvalho, D P; Fong, P; Guggino, W B; Morales, M

    2003-09-01

    Thyroid hormones has its main role in controlling metabolism, but it can also modulate extracellular fluid Volume (ECFV) through its action on the expression and activity of Na(+) transporters. Otherwise, chloride is the main anion in the ECFV and the influence of thyroid hormones in the regulation of chloride transporters is not yet understood. In this work, we studied the effect of thyroid hormones in the expression of ClC-2, a cell Volume-, pH- and voltage-sensitive Cl(-) channel, in rat kidney. To analyze the modulation of ClC-2 gene expression by thyroid hormones, we used hypothyroid (Hypo) rats with or without thyroxine (T(4)) replacement and hyperthyroid (Hyper) rats as our experimental models. Total RNA was isolated and the expression of ClC-2 mRNA was evaluated by a ribonuclease protection assay, and/or semi-quantitative RT-PCR. Renal ClC-2 expression decreased in Hypo rats and increased in Hyper rats. In addition, semi-quantitative RT-PCR of different nephron segments showed that these changes were due exclusively to the modulation of ClC-2 mRNA expression by thyroid hormone in convoluted and straight proximal tubules. To investigate whether thyroid hormones action was direct or indirect, renal proximal tubule primary culture cells were prepared and subjected to different T(4) concentrations. ClC-2 mRNA expression was increased by T(4) in a dose-dependent fashion, as analyzed by RT-PCR. Western blotting demonstrated that ClC-2 protein expression followed the same profile of mRNA expression.

  14. β-Pix directs collective migration of anterior visceral endoderm cells in the early mouse embryo.

    Science.gov (United States)

    Omelchenko, Tatiana; Rabadan, M Angeles; Hernández-Martínez, Rocío; Grego-Bessa, Joaquim; Anderson, Kathryn V; Hall, Alan

    2014-12-15

    Collective epithelial migration is important throughout embryonic development. The underlying mechanisms are poorly understood but likely involve spatially localized activation of Rho GTPases. We previously reported that Rac1 is essential for generating the protrusive activity that drives the collective migration of anterior visceral endoderm (AVE) cells in the early mouse embryo. To identify potential regulators of Rac1, we first performed an RNAi screen of Rho family exchange factors (guanine nucleotide exchange factor [GEF]) in an in vitro collective epithelial migration assay and identified β-Pix. Genetic deletion of β-Pix in mice disrupts collective AVE migration, while high-resolution live imaging revealed that this is associated with randomly directed protrusive activity. We conclude that β-Pix controls the spatial localization of Rac1 activity to drive collective AVE migration at a critical stage in mouse development.

  15. Electron-collecting oxide layers in inverted polymer solar cells via oxidation of thermally evaporated titanium

    Science.gov (United States)

    Zampetti, A.; Salamandra, L.; Brunetti, F.; Reale, A.; Di Carlo, A.; Brown, T. M.

    2016-10-01

    A simple and intuitive deposition technique is discussed to obtain titanium oxide used as an electron collecting layer in polymer solar cells based on the thermal evaporation of pristine titanium and further thermal treatment to convert the metal in oxide. Since the degradation of indium-doped tin oxide at high temperatures is an issue, we demonstrate that the combination of glass/fluorine tin oxide and high temperatures represents a promising approach in the fabrication of inverted polymer solar cells with such a titanium oxide electron collecting layer.

  16. Microbial Culture Collection (MCC) and International Depositary Authority (IDA) at National Centre for Cell Science, Pune.

    Science.gov (United States)

    Sharma, Avinash; Shouche, Yogesh

    2014-06-01

    Culture collections are valuable resources for the sustainable use of microbial diversity and its conservation. Advances in biotechnology have further increased their importance and some of these have been recognized as International Depositary Authority (IDA) for the deposition of patent cultures. Microbial Culture Collection at National Centre for Cell Science was established by the Department of Biotechnology, Government of India is country's newest culture collection with largest holdings. It is recognized as an IDA under the Budapest Treaty and Designated National Repository under the Biodiversity Act 2002. This article describes its various service related activities.

  17. CCR7 and IRF4-dependent dendritic cells regulate lymphatic collecting vessel permeability.

    Science.gov (United States)

    Ivanov, Stoyan; Scallan, Joshua P; Kim, Ki-Wook; Werth, Kathrin; Johnson, Michael W; Saunders, Brian T; Wang, Peter L; Kuan, Emma L; Straub, Adam C; Ouhachi, Melissa; Weinstein, Erica G; Williams, Jesse W; Briseño, Carlos; Colonna, Marco; Isakson, Brant E; Gautier, Emmanuel L; Förster, Reinhold; Davis, Michael J; Zinselmeyer, Bernd H; Randolph, Gwendalyn J

    2016-04-01

    Lymphatic collecting vessels direct lymph into and from lymph nodes (LNs) and can become hyperpermeable as the result of a previous infection. Enhanced permeability has been implicated in compromised immunity due to reduced flow of lymph and immune cells to LNs, which are the primary site of antigen presentation to T cells. Presently, very little is known about the molecular signals that affect lymphatic collecting vessel permeability. Here, we have shown that lymphatic collecting vessel permeability is controlled by CCR7 and that the chronic hyperpermeability of collecting vessels observed in Ccr7-/- mice is followed by vessel fibrosis. Reexpression of CCR7 in DCs, however, was sufficient to reverse the development of such fibrosis. IFN regulatory factor 4-positive (IRF4+) DCs constitutively interacted with collecting lymphatics, and selective ablation of this DC subset in Cd11c-Cre Irf4fl/fl mice also rendered lymphatic collecting vessels hyperpermeable and fibrotic. Together, our data reveal that CCR7 plays multifaceted roles in regulating collecting vessel permeability and fibrosis, with one of the key players being IRF4-dependent DCs.

  18. Dynamic tensile forces drive collective cell migration through three-dimensional extracellular matrices

    Science.gov (United States)

    Gjorevski, Nikolce; S. Piotrowski, Alexandra; Varner, Victor D.; Nelson, Celeste M.

    2015-01-01

    Collective cell migration drives tissue remodeling during development, wound repair, and metastatic invasion. The physical mechanisms by which cells move cohesively through dense three-dimensional (3D) extracellular matrix (ECM) remain incompletely understood. Here, we show directly that migration of multicellular cohorts through collagenous matrices occurs via a dynamic pulling mechanism, the nature of which had only been inferred previously in 3D. Tensile forces increase at the invasive front of cohorts, serving a physical, propelling role as well as a regulatory one by conditioning the cells and matrix for further extension. These forces elicit mechanosensitive signaling within the leading edge and align the ECM, creating microtracks conducive to further migration. Moreover, cell movements are highly correlated and in phase with ECM deformations. Migrating cohorts use spatially localized, long-range forces and consequent matrix alignment to navigate through the ECM. These results suggest biophysical forces are critical for 3D collective migration. PMID:26165921

  19. Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: role of Aquaporin-2.

    Science.gov (United States)

    Rivarola, Valeria; Flamenco, Pilar; Melamud, Luciana; Galizia, Luciano; Ford, Paula; Capurro, Claudia

    2010-08-01

    Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes.

  20. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

    OpenAIRE

    Supatto, Willy; McMahon, Amy; Fraser, Scott E.; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multiphoton microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and progr...

  1. The Cell Collective: Toward an open and collaborative approach to systems biology

    Directory of Open Access Journals (Sweden)

    Helikar Tomáš

    2012-08-01

    Full Text Available Abstract Background Despite decades of new discoveries in biomedical research, the overwhelming complexity of cells has been a significant barrier to a fundamental understanding of how cells work as a whole. As such, the holistic study of biochemical pathways requires computer modeling. Due to the complexity of cells, it is not feasible for one person or group to model the cell in its entirety. Results The Cell Collective is a platform that allows the world-wide scientific community to create these models collectively. Its interface enables users to build and use models without specifying any mathematical equations or computer code - addressing one of the major hurdles with computational research. In addition, this platform allows scientists to simulate and analyze the models in real-time on the web, including the ability to simulate loss/gain of function and test what-if scenarios in real time. Conclusions The Cell Collective is a web-based platform that enables laboratory scientists from across the globe to collaboratively build large-scale models of various biological processes, and simulate/analyze them in real time. In this manuscript, we show examples of its application to a large-scale model of signal transduction.

  2. Vitality of Enterococcus faecalis inside dentinal tubules after five root canal disinfection methods

    Directory of Open Access Journals (Sweden)

    Niranjan Ashok Vatkar

    2016-01-01

    Conclusion: E. faecalis has the ability to colonize inside dentinal tubules to a depth of >1000 μm. In contrast to conventional irrigants, both Nd: YAG and diode lasers were effective in eliminating the vitality of E. faecalis. NS, NaOCl, and CHX showed viable bacteria remaining in dentinal tubules.

  3. Mathematical study on robust tissue pattern formation in growing epididymal tubule.

    Science.gov (United States)

    Hirashima, Tsuyoshi

    2016-10-21

    Tissue pattern formation during development is a reproducible morphogenetic process organized by a series of kinetic cellular activities, leading to the building of functional and stable organs. Recent studies focusing on mechanical aspects have revealed physical mechanisms on how the cellular activities contribute to the formation of reproducible tissue patterns; however, the understanding for what factors achieve the reproducibility of such patterning and how it occurs is far from complete. Here, I focus on a tube pattern formation during murine epididymal development, and show that two factors influencing physical design for the patterning, the proliferative zone within the tubule and the viscosity of tissues surrounding to the tubule, control the reproducibility of epididymal tubule pattern, using a mathematical model based on experimental data. Extensive numerical simulation of the simple mathematical model revealed that a spatially localized proliferative zone within the tubule, observed in experiments, results in more reproducible tubule pattern. Moreover, I found that the viscosity of tissues surrounding to the tubule imposes a trade-off regarding pattern reproducibility and spatial accuracy relating to the region where the tubule pattern is formed. This indicates an existence of optimality in material properties of tissues for the robust patterning of epididymal tubule. The results obtained by numerical analysis based on experimental observations provide a general insight on how physical design realizes robust tissue pattern formation.

  4. The Hippo pathway polarizes the actin cytoskeleton during collective migration of Drosophila border cells.

    Science.gov (United States)

    Lucas, Eliana P; Khanal, Ichha; Gaspar, Pedro; Fletcher, Georgina C; Polesello, Cedric; Tapon, Nicolas; Thompson, Barry J

    2013-06-10

    Collective migration of Drosophila border cells depends on a dynamic actin cytoskeleton that is highly polarized such that it concentrates around the outer rim of the migrating cluster of cells. How the actin cytoskeleton becomes polarized in these cells to enable collective movement remains unknown. Here we show that the Hippo signaling pathway links determinants of cell polarity to polarization of the actin cytoskeleton in border cells. Upstream Hippo pathway components localize to contacts between border cells inside the cluster and signal through the Hippo and Warts kinases to polarize actin and promote border cell migration. Phosphorylation of the transcriptional coactivator Yorkie (Yki)/YAP by Warts does not mediate the function of this pathway in promoting border cell migration, but rather provides negative feedback to limit the speed of migration. Instead, Warts phosphorylates and inhibits the actin regulator Ena to activate F-actin Capping protein activity on inner membranes and thereby restricts F-actin polymerization mainly to the outer rim of the migrating cluster.

  5. New wrinkling substrate assay reveals traction force fields of leader and follower cells undergoing collective migration.

    Science.gov (United States)

    Yokoyama, Sho; Matsui, Tsubasa S; Deguchi, Shinji

    2017-01-22

    Physical forces play crucial roles in coordinating collective migration of epithelial cells, but details of such force-related phenomena remain unclear partly due to the lack of robust methodologies to probe the underlying force fields. Here we develop a method for fabricating silicone substrates that detect cellular traction forces with a high sensitivity. Specifically, a silicone elastomer is exposed to oxygen plasma under heating. Removal of the heat shrinks the substrate so as to reduce its critical buckling strain in a spatially uniform manner. Thus, even small cellular traction forces can be visualized as micro-wrinkles that are reversibly emerged on the substrate in a direction orthogonal to the applied forces. Using this technique, we show that so-called leader cells in MDCK-II cell clusters exert significant magnitudes of traction forces distinct from those of follower cells. We reveal that the direction of traction forces is highly correlated with the long axis of the local, individual cells within clusters. These results suggest that the force fields in collective migration of MDCK-II cells are predominantly determined locally at individual cell scale rather than globally at the whole cell cluster scale. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Regional variation in root dentinal tubule infection by Streptococcus gordonii.

    Science.gov (United States)

    Love, R M

    1996-06-01

    The purpose of this study was to investigate the pattern of bacterial invasion of dentinal tubules at different regions in human roots. Specimens were obtained from single-rooted teeth that had their root canals prepared in a standard manner. Roots were then sectioned longitudinally through the canals and the resulting specimens chemically treated to remove the smear layers. Specimens were immersed in a suspension of Streptococcus gordonii for 3 weeks and then prepared for histological analysis. Sections from the cervical, midroot, and apical areas were examined. The pattern of bacterial infection of the cervical and midroot areas was similar, characterized as a heavy infection with bacteria penetrating as deep as 200 microns. Invasion of the apical dentin was significantly different, with a mild infection and maximum penetration of 60 microns.

  7. Static analysis of masonry kilns built with fictile tubules bricks

    Science.gov (United States)

    Olivito, Renato S.; Scuro, Carmelo; Codispoti, Rosamaria

    2016-12-01

    Industrial archeology is a branch that studies all the testimony (tangible and intangible, direct and indirect) related to the process of industrialization since its origins. This technical field is based on an interdisciplinary approach, it has the task of deepening the story, understanding the technological development made by man over the centuries. The present work focused attention on the study and analysis of a masonry kiln, built with the technique of hollow clay fictile tubules. The study, in particular, has been carried out analyzing the stress state caused by the wind on the structure. The kiln is constituted by a particular geometric configuration that develops in height due to the presence of chimney over the dome.

  8. Reversible effects of acute hypertension on proximal tubule sodium transporters

    DEFF Research Database (Denmark)

    Zhang, Y; Magyar, C E; Norian, J M

    1998-01-01

    Acute hypertension provokes a rapid decrease in proximal tubule sodium reabsorption with a decrease in basolateral membrane sodium-potassium-ATPase activity and an increase in the density of membranes containing apical membrane sodium/hydrogen exchangers (NHE3) [Y. Zhang, A. K. Mircheff, C. B....... Renal cortex lysate was fractionated on sorbitol gradients. Basolateral membrane sodium-potassium-ATPase activity (but not subunit immunoreactivity) decreased one-third to one-half after BP was elevated and recovered after BP was normalized. After BP was elevated, 55% of the apical NHE3 immunoreactivity......, smaller fractions of sodium-phosphate cotransporter immunoreactivity, and apical alkaline phosphatase and dipeptidyl-peptidase redistributed to membranes of higher density enriched in markers of the intermicrovillar cleft (megalin) and endosomes (Rab 4 and Rab 5), whereas density distributions...

  9. Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

    NARCIS (Netherlands)

    Engelse, M.A.; Laurens, N.; Verloop, R.E.; Koolwijk, P.; Hinsbergh, V.W.M. van

    2008-01-01

    The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expr

  10. Recurrent infarction of sphenoid bone with subperiosteal collection in a child with sickle cell disease.

    Science.gov (United States)

    Alsuhaibani, Adel H; Marzouk, Mohammed Abu

    2011-01-01

    Infarction of the orbital bone in patients with sickle cell disease is very rare. The authors report a young boy who presented twice with marked acute proptosis and eyelid swelling of the right eye resulting from infarction in the greater wing of the sphenoid bone accompanied by an orbital subperiosteal collection. The time interval between the 2 attacks was 3 years.

  11. Mechanotransduction in mechanically coupled pulsating cells: transition to collective constriction and mesoderm invagination simulation

    Science.gov (United States)

    Driquez, Benjamin; Bouclet, Adrien; Farge, Emmanuel

    2011-12-01

    Embryonic differentiation and morphogenesis require the coordination of the cascades of gene product expression with the morphogenetic sequence of development. The influence of mechanical deformations driven by morphogenetic movements on biochemical activities was recently revealed by the existence of mechanotransduction processes in development, involving both gene transcription and protein behaviour. In the early Drosophila embryo, apical stabilization of Myosin-II leading to mesoderm invagination at the onset of gastrulation was proposed to be triggered in response to the activation of the Fog mechanotransduction pathway by the Snail-dependent active mechanical oscillations of cell apex sizes. Here we simulate the mesoderm as mechanically coupled cells, with pulsatile forces of constriction at the cell level mimicking Snail-dependent active fluctuations of apexes. We define a critical apex diameter triggering active constriction that mimics the activation of the Fog mechanotransduction pathway leading to cell constriction. We find that collective movements trigger the dynamical transition to constriction predicting the experimental dynamics of mesoderm cell apex size decrease with a modulus of contractility four times higher than the passive modulus of elastic deformation of the cells. The contraction wave is activated in a pulsation frequency-dependent process, and propagates at multicellular scales through local cell-cell mechanical interactions. By reproducing the pattern of Snail and Fog gene product protein expression in a simulation of ventral cells, the model phenocopies the pattern of Myo-II apical stabilization, and the dynamic pattern of constriction that initiates along a central sub-domain of the mesoderm. We propose that multicellular mechanical collective effects couple with mechanotransduction biochemical mechanisms to trigger the transition of collective coordinated constriction, through a mechano-genetic process ensuring efficient and regular

  12. Mechanotransduction in mechanically coupled pulsating cells: transition to collective constriction and mesoderm invagination simulation.

    Science.gov (United States)

    Driquez, Benjamin; Bouclet, Adrien; Farge, Emmanuel

    2011-12-01

    Embryonic differentiation and morphogenesis require the coordination of the cascades of gene product expression with the morphogenetic sequence of development. The influence of mechanical deformations driven by morphogenetic movements on biochemical activities was recently revealed by the existence of mechanotransduction processes in development, involving both gene transcription and protein behaviour. In the early Drosophila embryo, apical stabilization of Myosin-II leading to mesoderm invagination at the onset of gastrulation was proposed to be triggered in response to the activation of the Fog mechanotransduction pathway by the Snail-dependent active mechanical oscillations of cell apex sizes. Here we simulate the mesoderm as mechanically coupled cells, with pulsatile forces of constriction at the cell level mimicking Snail-dependent active fluctuations of apexes. We define a critical apex diameter triggering active constriction that mimics the activation of the Fog mechanotransduction pathway leading to cell constriction. We find that collective movements trigger the dynamical transition to constriction predicting the experimental dynamics of mesoderm cell apex size decrease with a modulus of contractility four times higher than the passive modulus of elastic deformation of the cells. The contraction wave is activated in a pulsation frequency-dependent process, and propagates at multicellular scales through local cell-cell mechanical interactions. By reproducing the pattern of Snail and Fog gene product protein expression in a simulation of ventral cells, the model phenocopies the pattern of Myo-II apical stabilization, and the dynamic pattern of constriction that initiates along a central sub-domain of the mesoderm. We propose that multicellular mechanical collective effects couple with mechanotransduction biochemical mechanisms to trigger the transition of collective coordinated constriction, through a mechano-genetic process ensuring efficient and regular

  13. Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics.

    Science.gov (United States)

    Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic; Jennings, Paul

    2015-12-25

    Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 μM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Changes of androgen receptor expression in stages VII-VIII seminiferous tubules of rat testis after exposure to methamphetamine

    Directory of Open Access Journals (Sweden)

    Sutita Nudmamud-Thanoi

    2016-06-01

    Full Text Available Methamphetamine (METH is toxic not only to the central nervous system but also to the reproductive system. This study aimed to investigate the effect of METH administration on androgen receptor (AR expression in the seminiferous tubules (stages VII-VIII and Leydig cells. 24 Sprague-Dawley rats were equally divided into 4 groups; control, acute doseMETH binge (AB-METH, escalating dose-METH (ED-METH and escalating dose-METH binge (ED-METH binge groups. The expressions of AR were examined using immunohistochemical staining. AR expressions in round and elongated spermatids showed significant decreases in ED-METH binge and AB-METH groups. In Leydig’s cells, AR expressions were significantly decreased in both ED-METH and ED-METH binge groups. These results could reflect the effect of METH on the expressions of AR in both round and elongated spermatids of stages VII-VIII seminiferous tubule and Leydig’s cells. Decreases of AR expressions in those cells of METH-treated animals may interrupt spermatogenesis at these stages.

  15. TLR4 facilitates translocation of bacteria across renal collecting duct cells.

    Science.gov (United States)

    Chassin, Cécilia; Vimont, Sophie; Cluzeaud, Françoise; Bens, Marcelle; Goujon, Jean-Michel; Fernandez, Béatrice; Hertig, Alexandre; Rondeau, Eric; Arlet, Guillaume; Hornef, Mathias W; Vandewalle, Alain

    2008-12-01

    Uropathogenic Escherichia coli (UPEC) are the most frequent causes of urinary tract infections and pyelonephritis. Renal medullary collecting duct (MCD) cells are the intrarenal site to which UPEC strains prefer to adhere and initiate an inflammatory response, but the ability of UPEC strains to translocate across impermeant MCD cells has not been demonstrated definitively. Here, several UPEC strains adhered to the apical surface and translocated across confluent murine inner MCD cells grown on filters. UPEC strains expressing cytolytic and vacuolating cytotoxins disrupted the integrity of cell layers, whereas noncytolytic UPEC strains passed through the cell layers without altering tight junctions. Apical-to-basal transcellular translocation was dramatically reduced after extinction of Toll-like receptor 4 (TLR4) and the lipid raft marker caveolin-1 by small interfering RNA. Furthermore, disruption of lipid raft integrity by filipin III and methyl-beta-cyclodextrin significantly reduced both the transcellular translocation of UPEC across murine inner MCD cell layers and the stimulation of proinflammatory mediators. Bacterial translocation was also significantly reduced in primary cultures of TLR4-deficient mouse MCD cells compared with MCD cells from wild-type mice. Benzyl alcohol, an anesthetic that enhances membrane fluidity, favored the recruitment of caveolin-1 in lipid rafts and increased the translocation of UPEC across cultured TLR4-deficient MCD cells. These findings demonstrate that the transcellular translocation of UPEC strains across impermeant layers of MCD cells may occur through lipid rafts via a TLR4-facilitated process.

  16. Changes of androgen receptor expression in stages VII-VIII seminiferous tubules of rat testis after exposure to methamphetamine

    OpenAIRE

    Sutita Nudmamud-Thanoi; Nareelak Tangsrisakda; Samur Thanoi

    2016-01-01

    Methamphetamine (METH) is toxic not only to the central nervous system but also to the reproductive system. This study aimed to investigate the effect of METH administration on androgen receptor (AR) expression in the seminiferous tubules (stages VII-VIII) and Leydig cells. 24 Sprague-Dawley rats were equally divided into 4 groups; control, acute doseMETH binge (AB-METH), escalating dose-METH (ED-METH) and escalating dose-METH binge (ED-METH binge) groups. The expressions of AR we...

  17. Cell Phones to Collect Pregnancy Data From Remote Areas in Liberia

    Science.gov (United States)

    Lori, Jody R.; Munro, Michelle L.; Boyd, Carol J.; Andreatta, Pamela

    2012-01-01

    Purpose To report findings on knowledge and skill acquisition following a 3-day training session in the use of short message service (SMS) texting with non- and low-literacy traditional midwives. Design A pre- and post-test study design was used to assess knowledge and skills acquisition with 99 traditional midwives on the use of SMS texting for real-time, remote data collection in rural Liberia, West Africa. Methods Paired sample t-tests were conducted to establish if overall mean scores varied significantly from pre-test to immediate post-test. Analysis of variance was used to compare means across groups. The nonparametric McNemar’s test was used to determine significant differences between the pre-test and post-test values of each individual step involved in SMS texting. Pearson’s chi-square test of independence was used to examine the association between ownership of cell phones within a family and achievement of the seven tasks. Findings The mean increase in cell phone knowledge scores was 3.67, with a 95% confidence interval ranging from 3.39 to 3.95. Participants with a cell phone in the family did significantly better on three of the seven tasks in the pre-test: “turns cell on without help” (χ2(1) = 9.15, p = .003); “identifies cell phone coverage” (χ2(1) = 5.37, p = .024); and “identifies cell phone is charged” (χ2(1) = 4.40, p = .042). Conclusions A 3-day cell phone training session with low- and nonliterate traditional midwives in rural Liberia improved their ability to use mobile technology for SMS texting. Clinical Relevance Mobile technology can improve data collection accessibility and be used for numerous healthcare and public health issues. Cell phone accessibility holds great promise for collecting health data in low-resource areas of the world. PMID:22672157

  18. Proteomic profiling of nuclear fractions from native renal inner medullary collecting duct cells.

    Science.gov (United States)

    Pickering, Christina M; Grady, Cameron; Medvar, Barbara; Emamian, Milad; Sandoval, Pablo C; Zhao, Yue; Yang, Chin-Rang; Jung, Hyun Jun; Chou, Chung-Lin; Knepper, Mark A

    2016-02-01

    The control of renal water excretion occurs in part by regulation of transcription in response to vasopressin in cells of the collecting duct. A systems biology-based approach to understanding transcriptional control in renal collecting duct cells depends on knowledge of what transcription factors and other regulatory proteins are present in the cells' nuclei. The goal of this article is to report comprehensive proteomic profiling of cellular fractions enriched in nuclear proteins from native inner medullary collecting duct (IMCD) cells of the rat. Multidimensional separation procedures and state-of-the art protein mass spectrometry produced 18 GB of spectral data that allowed the high-stringency identification of 5,048 proteins in nuclear pellet (NP) and nuclear extract (NE) fractions of biochemically isolated rat IMCD cells (URL: https://helixweb.nih.gov/ESBL/Database/IMCD_Nucleus/). The analysis identified 369 transcription factor proteins out of the 1,371 transcription factors coded by the rat genome. The analysis added 1,511 proteins to the recognized proteome of rat IMCD cells, now amounting to 8,290 unique proteins. Analysis of samples treated with the vasopressin analog dDAVP (1 nM for 30 min) or its vehicle revealed 99 proteins in the NP fraction and 88 proteins in the NE fraction with significant changes in spectral counts (Fisher exact test, P < 0.005). Among those altered by vasopressin were seven distinct histone proteins, all of which showed decreased abundance in the NP fraction, consistent with a possible effect of vasopressin to induce chromatin remodeling. The results provide a data resource for future studies of vasopressin-mediated transcriptional regulation in the renal collecting duct.

  19. Mean-cluster approach indicates cell sorting time scales are determined by collective dynamics

    Science.gov (United States)

    Beatrici, Carine P.; de Almeida, Rita M. C.; Brunnet, Leonardo G.

    2017-03-01

    Cell migration is essential to cell segregation, playing a central role in tissue formation, wound healing, and tumor evolution. Considering random mixtures of two cell types, it is still not clear which cell characteristics define clustering time scales. The mass of diffusing clusters merging with one another is expected to grow as td /d +2 when the diffusion constant scales with the inverse of the cluster mass. Cell segregation experiments deviate from that behavior. Explanations for that could arise from specific microscopic mechanisms or from collective effects, typical of active matter. Here we consider a power law connecting diffusion constant and cluster mass to propose an analytic approach to model cell segregation where we explicitly take into account finite-size corrections. The results are compared with active matter model simulations and experiments available in the literature. To investigate the role played by different mechanisms we considered different hypotheses describing cell-cell interaction: differential adhesion hypothesis and different velocities hypothesis. We find that the simulations yield normal diffusion for long time intervals. Analytic and simulation results show that (i) cluster evolution clearly tends to a scaling regime, disrupted only at finite-size limits; (ii) cluster diffusion is greatly enhanced by cell collective behavior, such that for high enough tendency to follow the neighbors, cluster diffusion may become independent of cluster size; (iii) the scaling exponent for cluster growth depends only on the mass-diffusion relation, not on the detailed local segregation mechanism. These results apply for active matter systems in general and, in particular, the mechanisms found underlying the increase in cell sorting speed certainly have deep implications in biological evolution as a selection mechanism.

  20. Stages of Renal Cell Cancer

    Science.gov (United States)

    ... cell cancer is a disease in which malignant (cancer) cells form in tubules of the kidney. Renal cell ... diagnosed, tests are done to find out if cancer cells have spread within the kidney or to other ...

  1. Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium.

    Science.gov (United States)

    Dalle Nogare, Damian; Somers, Katherine; Rao, Swetha; Matsuda, Miho; Reichman-Fried, Michal; Raz, Erez; Chitnis, Ajay B

    2014-08-01

    Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells. © 2014. Published by The Company of Biologists Ltd.

  2. Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium

    Science.gov (United States)

    Dalle Nogare, Damian; Somers, Katherine; Rao, Swetha; Matsuda, Miho; Reichman-Fried, Michal; Raz, Erez; Chitnis, Ajay B.

    2014-01-01

    Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells. PMID:25063456

  3. In Situ Single Photon Confocal Imaging of Cardiomyocyte T-tubule System from Langendorff-Perfused Hearts

    Directory of Open Access Journals (Sweden)

    Biyi eChen

    2015-05-01

    Full Text Available Transverse tubules (T-tubules are orderly invaginations of the sarcolemma in mammalian cardiomyocytes. The integrity of T-tubule architecture is critical for cardiac excitation-contraction coupling function. T-tubule remodeling is recognized as a key player in cardiac dysfunction. Early studies on T-tubule structure were based on electron microscopy, which uncovered important information about the T-tubule architecture. The advent of fluorescent membrane probes allowed the application of confocal microscopy to investigations of T-tubule structure. Studies have now been extended beyond single cardiomyocytes to examine the T-tubule network in intact hearts through in situ confocal imaging of Langendorff-perfused hearts. This technique has allowed visualization of T-tubule organization in their natural habitat, avoiding the damage induced by isolation of cardiomyocytes. Additionally, it is possible to obtain T-tubule images in different subepicardial regions in a single intact heart. We review how this state-of-the-art imaging technique has provided important mechanistic insights into maturation of T-tubules in developing hearts and defined the role of T-tubule remodeling in development and progression of heart failure.

  4. AKT primes snail-induced EMT concomitantly with the collective migration of squamous cell carcinoma cells.

    Science.gov (United States)

    Okui, Gaku; Tobiume, Kei; Rizqiawan, Andra; Yamamoto, Kazuhiro; Shigeishi, Hideo; Ono, Shigehiro; Higashikawa, Koichiro; Kamata, Nobuyuki

    2013-09-01

    In this study, we found that wounding of a confluent monolayer of squamous cell carcinoma (SCC) cells induced epithelial-mesenchymal transition (EMT) specifically at the edge of the wound. This process required the combined stimulation of TGFβ, TNFα, and PDGF-D. Such a combined cytokine treatment of confluent monolayers of the cells upregulated the expression levels of Snail and Slug via PI3K. The PI3K downstream effector, AKT, was dispensable for the upregulation of Snail and Slug, but essential for enabling EMT in response to upregulation of Snail and Slug. Copyright © 2013 Wiley Periodicals, Inc.

  5. Carrier collection losses in interface passivated amorphous silicon thin-film solar cells

    Science.gov (United States)

    Neumüller, A.; Bereznev, S.; Ewert, M.; Volobujeva, O.; Sergeev, O.; Falta, J.; Vehse, M.; Agert, C.

    2016-07-01

    In silicon thin-film solar cells the interface between the i- and p-layer is the most critical. In the case of back diffusion of photogenerated minority carriers to the i/p-interface, recombination occurs mainly on the defect states at the interface. To suppress this effect and to reduce recombination losses, hydrogen plasma treatment (HPT) is usually applied. As an alternative to using state of the art HPT we apply an argon plasma treatment (APT) before the p-layer deposition in n-i-p solar cells. To study the effect of APT, several investigations were applied to compare the results with HPT and no plasma treatment at the interface. Carrier collection losses in resulting solar cells were examined with spectral response measurements with and without bias voltage. To investigate single layers, surface photovoltage and X-ray photoelectron spectroscopy (XPS) measurements were conducted. The results with APT at the i/p-interface show a beneficial contribution to the carrier collection compared with HPT and no plasma treatment. Therefore, it can be concluded that APT reduces the recombination centers at the interface. Further, we demonstrate that carrier collection losses of thin-film solar cells are significantly lower with APT.

  6. Leader cells regulate collective cell migration via Rac activation in the downstream signaling of integrin β1 and PI3K.

    Science.gov (United States)

    Yamaguchi, Naoya; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2015-01-07

    Collective cell migration plays a crucial role in several biological processes, such as embryonic development, wound healing, and cancer metastasis. Here, we focused on collectively migrating Madin-Darby Canine Kidney (MDCK) epithelial cells that follow a leader cell on a collagen gel to clarify the mechanism of collective cell migration. First, we removed a leader cell from the migrating collective with a micromanipulator. This then caused disruption of the cohesive migration of cells that followed in movement, called "follower" cells, which showed the importance of leader cells. Next, we observed localization of active Rac, integrin β1, and PI3K. These molecules were clearly localized in the leading edge of leader cells, but not in follower cells. Live cell imaging using active Rac and active PI3K indicators was performed to elucidate the relationship between Rac, integrin β1, and PI3K. Finally, we demonstrated that the inhibition of these molecules resulted in the disruption of collective migration. Our findings not only demonstrated the significance of a leader cell in collective cell migration, but also showed that Rac, integrin β1, and PI3K are upregulated in leader cells and drive collective cell migration.

  7. Cell jamming: Collective invasion of mesenchymal tumor cells imposed by tissue confinement

    NARCIS (Netherlands)

    Haeger, A.; Krause, M.; Wolf, K. van der; Friedl, P.

    2014-01-01

    BACKGROUND: Cancer invasion is a multi-step process which coordinates interactions between tumor cells with mechanotransduction towards the surrounding matrix, resulting in distinct cancer invasion strategies. Defined by context, mesenchymal tumors, including melanoma and fibrosarcoma, develop eithe

  8. Comparison of the Fenwal Amicus and Fresenius Com.Tec cell separators for autologous peripheral blood progenitor cell collection.

    Science.gov (United States)

    Altuntas, Fevzi; Kocyigit, Ismail; Ozturk, Ahmet; Kaynar, Leylagul; Sari, Ismail; Oztekin, Mehmet; Solmaz, Musa; Eser, Bulent; Cetin, Mustafa; Unal, Ali

    2007-04-01

    Peripheral blood progenitor cells (PBPC) are commonly used as a stem cell source for autologous transplantation. This study was undertaken to evaluate blood cell separators with respect to separation results and content of the harvest. Forty autologous PBPC collections in patients with hematological malignancies were performed with either the Amicus or the COM.TEC cell separators. The median product volume was lower with the Amicus compared to the COM.TEC (125 mL vs. 300 mL; p COM.TEC (3.0 x 10(6) vs. 4.1 x 10(6); p = 0.129). There was a statistically higher mean volume of ACD used in collections on the Amicus compared to the COM.TEC (1040 +/- 241 mL vs. 868 +/- 176 mL; p = 0.019). There was a statistical difference in platelet (PLT) contamination of the products between the Amicus and the COM.TEC (0.3 x 10(11) vs. 1.1 x 10(11); p COM.TEC compared to the Amicus instruments (18.5% vs. 9.5%; p = 0.028). In conclusion, both instruments collected PBPCs efficiently. However, Amicus has the advantage of lower PLT contamination in the product, and less decrease in PB platelet count with lower product volume in autologous setting.

  9. ImageJ analysis of dentin tubule distribution in human teeth.

    Science.gov (United States)

    Williams, Casia; Wu, Yiching; Bowers, Doria F

    2015-08-01

    Mapping the distribution of dentin tubules is vital to understanding the structure-function relationship of dentin, an important indicator of tooth stability. This study compared the distances between and density of tubules in the external dentin located in the crown region of an adult human incisor and molar to determine if analysis could be conducted using light-level microscopy. Teeth were processed for routine histology, cut in cross-section, images captured using Advanced SPOT Program, and microstructure was analyzed using ImageJ (NIH). Intratubular (peritubular) dentin with or without odontoblast processes were observed and although incisor and molar images appeared visually similar, plot profile graphs differed. Distance-intervals between tubules in the incisor (5.45-7.67 μm) had an overall range of 2.22 μm and in the molar (7.43-8.42 μm) an overall range of 0.99 μm. While molar tubule distribution displayed a tighter overall range, there was a smaller distance between most incisor tubules. The average densities observed in incisors were 15,500 tubules/mm(2), compared with 20,100 tubules/mm(2) in molars. ImageJ analysis of prepared histology microscopic slides provides researchers with a rapid, inexpensive assessment tool when compared with advanced/ultrastructural methodologies. By combining routine histological processing and light microscopic observations followed by ImageJ analysis, tooth structure can be converted into numerical data and easily mastered by laboratory personnel.

  10. Modelling the effect of curvature on the collective behaviour of cells growing new tissue

    CERN Document Server

    Alias, Almie

    2016-01-01

    The growth of several biological tissues is known to be controlled in part by local geometrical features, such as the curvature of the tissue interface. This control leads to changes in tissue shape that in turn can affect the tissue's evolution. Understanding the cellular basis of this control is highly significant for bioscaffold tissue engineering, the evolution of bone microarchitecture, wound healing, and tumour growth. While previous models have proposed geometrical relationships between tissue growth and curvature, the role of cell density and cell vigor remains poorly understood. We propose a cell-based mathematical model of tissue growth to investigate the systematic influence of curvature on the collective crowding or spreading of tissue-synthesising cells induced by changes in local tissue surface area during the motion of the interface. Depending on the strength of diffusive damping, the model exhibits complex growth patterns such as undulating motion, efficient smoothing of irregularities, and th...

  11. Micro- and nano-tubules built from loosely and tightly rolled up thin sheets.

    Science.gov (United States)

    Losensky, Luisa; Goldenbogen, Björn; Holland, Gudrun; Laue, Michael; Petran, Anca; Liebscher, Jürgen; Scheidt, Holger A; Vogel, Alexander; Huster, Daniel; Klipp, Edda; Arbuzova, Anna

    2016-01-14

    Tubular structures built from amphiphilic molecules are of interest for nano-sensing, drug delivery, and structuring of oils. In this study, we characterized the tubules built in aqueous suspensions of a cholesteryl nucleoside conjugate, cholesterylaminouridine (CholAU) and phosphatidylcholines (PCs). In mixtures with unsaturated PCs having chain lengths comparable to the length of CholAU, two different types of tubular structures were observed; nano- and micro-tubules had average diameters in the ranges 50-300 nm and 2-3 μm, respectively. Using cryo scanning electron microscopy (cryo-SEM) we found that nano- and micro-tubules differed in their morphology: the nano-tubules were densely packed, whereas micro-tubules consisted of loosely rolled undulated lamellas. Atomic force microscopy (AFM) revealed that the nano-tubules were built from 4 to 5 nm thick CholAU-rich bilayers, which were in the crystalline state. Solid-state (2)H NMR spectroscopy also confirmed that about 25% of the total CholAU, being about the fraction of CholAU composing the tubules, formed the rigid crystalline phase. We found that CholAU/PC tubules can be functionalized by molecules inserted into lipid bilayers and fluorescently labeled PCs and lipophilic nucleic acids inserted spontaneously into the outer layer of the tubules. The tubular structures could be loaded and cross-linked, e.g. by DNA hybrids, and, therefore, are of interest for further development, e.g. as a depot scaffold for tissue regeneration.

  12. Role of AQP2 during apoptosis in cortical collecting duct cells.

    Science.gov (United States)

    Flamenco, Pilar; Galizia, Luciano; Rivarola, Valeria; Fernandez, Juan; Ford, Paula; Capurro, Claudia

    2009-04-01

    A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume decrease, due to the cellular outflow of potassium and chloride ions, followed by osmotically obliged water. In many cells, the ionic pathways triggered during the apoptotic volume decrease may be similar to that observed during a regulatory volume decrease response under hypotonic conditions. However, the pathways involved in water loss during apoptosis have been largely ignored. It was recently reported that in some systems this water movement is mediated via specific water channels (aquaporins). Nevertheless, it is important to identify whether this is a ubiquitous aspect of apoptosis as well as to define the mechanisms involved. The aim of the present work was to investigate the role of aquaporin-2 during apoptosis in renal-collecting duct cells. We evaluated the putative relationship between aquaporin-2 expression and the activation of the ionic pathways involved in the regulatory volume response. Apoptosis was induced by incubating cells with a hypertonic solution or with cycloheximide in two cortical collecting duct cell lines: one not expressing aquaporins and the other stably transfected with aquaporin-2. Typical features of apoptosis were evaluated with different approaches and the water permeability was measured by fluorescence videomicroscopy. Our results show that the rate of apoptosis is significantly increased in aquaporin-2 cells and it is linked to the rapid activation of volume-regulatory potassium and chloride channels. Furthermore, the water permeability of cells expressing aquaporin-2 was strongly reduced during the apoptotic process and it occurs before DNA degradation. These results let us propose that under apoptotic stimulation aquaporin-2 would act as a sensor leading to a co-ordinated activation of specific ionic channels for potassium and chloride efflux, resulting in both more rapid cell shrinkage and more rapid achievement of adequate levels of ions necessary to

  13. Defective planar cell polarity in polycystic kidney disease.

    Science.gov (United States)

    Fischer, Evelyne; Legue, Emilie; Doyen, Antonia; Nato, Faridabano; Nicolas, Jean-François; Torres, Vicente; Yaniv, Moshe; Pontoglio, Marco

    2006-01-01

    Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.

  14. Enhanced electron photoemission by collective lattice resonances in plasmonic nanoparticle-array photodetectors and solar cells

    CERN Document Server

    Zhukovsky, Sergei V; Uskov, Alexander V; Protsenko, Igor E; Lavrinenko, Andrei V

    2013-01-01

    We propose to use collective lattice resonances in plasmonic nanoparticle arrays to enhance photoelectron emission in Schottky-barrier photodetectors and solar cells. We show that the interaction of lattice resonances (the Rayleigh anomaly) and individual particle excitations (localized surface plasmon resonances) leads to stronger local field enhancement and significant increase of the photocurrent compared to the case when only individual particle excitations are present. The results can be used to design new photodetectors with highly selective, tunable spectral response, able to detect photons with the energy below the semiconductor bandgap, and to develop solar cells with increased efficiency.

  15. The spermatogonial stem cell niche

    NARCIS (Netherlands)

    D.G. de Rooij

    2009-01-01

    Spermatogonial stem cells (SSCs; A(s) spermatogonia) and their direct descendants (A(pr) and A(al) spermatogonia) are preferentially located in those areas of the seminiferous tubules that border on the interstitial tissue. Fewer of these cells are present in tubule areas directly bordering on anoth

  16. Quantitative phosphoproteomics in nuclei of vasopressin-sensitive renal collecting duct cells

    OpenAIRE

    Bolger, Steven J.; Hurtado, Patricia A. Gonzales; Hoffert, Jason D.; Saeed, Fahad; Pisitkun, Trairak; Knepper, Mark A.

    2012-01-01

    Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites...

  17. Hematologic assessment in pet rats, mice, hamsters, and gerbils: blood sample collection and blood cell identification.

    Science.gov (United States)

    Lindstrom, Nicole M; Moore, David M; Zimmerman, Kurt; Smith, Stephen A

    2015-01-01

    Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

  18. Talking about killing: Cell Phones, Collective Action, and Insurgent Violence in Iraq

    Science.gov (United States)

    2011-05-31

    technology, insurgency, collective action Jacob Shapiro, Nils Weidmann Harvard University Office of Sponsored Research 1350 Massachusetts Ave. Holyoke 727...at the district level changes as a result of the introduction of new cell phone towers. Second, using a novel identification strategy, we examine how... identification strategy, we examine how insurgent operation in the tower’s vicinity is affected by the introduction of coverage. Taken together, our

  19. Expression of lipases and lipid receptors in sperm storage tubules and possible role of fatty acids in sperm survival in the hen oviduct.

    Science.gov (United States)

    Huang, A; Isobe, N; Obitsu, T; Yoshimura, Y

    2016-04-15

    The aim of this study was to determine the role of fatty acids for sperm survival in the sperm storage tubules (SSTs) of the hen oviduct. The mucosa tissues of uterovaginal junction (UVJ) of White Leghorn laying hens with or without artificial insemination using semen from Barred Plymouth Rock roosters were collected. The lipid density in the epithelium of UVJ and SST was analyzed by Sudan black B staining. The expressions of genes encoding lipid receptors and lipases were assayed by polymerase chain reaction in UVJ mucosa and SST cells isolated by laser microdissection. Fatty acid composition was analyzed by gas chromatography, and sperm were cultured with or without the identified predominant fatty acids for 24 hours to examine their effect on sperm viability. The lipid droplets were localized in the epithelium of UVJ mucosa and SSTs. The expression of genes encoding very low-density lipoprotein receptor(VLDLR), low-density lipoprotein receptor (LDLR), and fatty acid translocase (FAT/CD36) were found in SST cells. Expression of genes encoding endothelial lipase (EL), lipase H (LIPH), adipose triglyceride lipase (ATGL), and lipoprotein lipase (LPL) were found in UVJ. In contrast, only ATGL was found in SST cells, and its expression was significantly upregulated after artificial insemination. In UVJ mucosal tissues, five fatty acids, namely myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1n9), and linoleic acid (C18:2n6), were identified as predominant fatty acids. The viability of sperm cultured with 1 mM oleic acid or linoleic acid was significantly higher than the sperm in the control culture without fatty acids. These results suggest that lipids in the SST cells may be degraded by ATGL, and fatty acids including oleic acid and linoleic acid may be released into the SST lumen to support sperm survival.

  20. Effects of Collective Histone State Dynamics on Epigenetic Landscape and Kinetics of Cell Reprogramming

    Science.gov (United States)

    Ashwin, S. S.; Sasai, Masaki

    2015-11-01

    Cell reprogramming is a process of transitions from differentiated to pluripotent cell states via transient intermediate states. Within the epigenetic landscape framework, such a process is regarded as a sequence of transitions among basins on the landscape; therefore, theoretical construction of a model landscape which exhibits experimentally consistent dynamics can provide clues to understanding epigenetic mechanism of reprogramming. We propose a minimal gene-network model of the landscape, in which each gene is regulated by an integrated mechanism of transcription-factor binding/unbinding and the collective chemical modification of histones. We show that the slow collective variation of many histones around each gene locus alters topology of the landscape and significantly affects transition dynamics between basins. Differentiation and reprogramming follow different transition pathways on the calculated landscape, which should be verified experimentally via single-cell pursuit of the reprogramming process. Effects of modulation in collective histone state kinetics on transition dynamics and pathway are examined in search for an efficient protocol of reprogramming.

  1. Collective interaction of microscale matters in natural analogy: human cancer cells vs. microspheres

    Science.gov (United States)

    Ahn, Sungsook; Lee, Sang Joon; Postech Team

    2014-11-01

    Collective behaviors have been considered both in living and lifeless things as a natural phenomenon. During the ordering process, a sudden and spontaneous transition is typically generated between an order and a disorder according to the population density of interacting elements. In a cellular level collective behavior, the cells are distributed in the characteristic patterns according to the population density and the mutual interaction of the individual cells undergo density-dependent diffusive motion. On the other hand, density-controlled surface-modified hollow microsphere suspension induces an overpopulation via buoyancy which provides a driving force to induce an assembly. The collective behaviors of the cells and microspheres in a designed liquid medium are explained in terms of the deviation from the interparticle distance distribution and the induced strength to organize the particle position in a specific distance range. as a result, microscale particulate matters exhibit high resemblance in their pair correlation and dynamical heterogeneity in the intermediate range between a single individual and an agglomerate. Therefore, it is suggested that biological systems are analogically explained to be dominated by physically interactive aspects.

  2. Endothelial directed collective migration depends on substrate stiffness via localized myosin contractility and cell-matrix interactions.

    Science.gov (United States)

    Canver, Adam Charles; Ngo, Olivia; Urbano, Rebecca Lownes; Clyne, Alisa Morss

    2016-05-24

    Macrovascular endothelial injury, which may be caused by percutaneous intervention, requires endothelial cell directed collective migration to restore an intact endothelial monolayer. While interventions are often performed in arteries stiffened by cardiovascular disease, the effect of substrate stiffness on endothelial cell collective migration has not been examined. We studied porcine aortic endothelial cell directed collective migration using a modified cage assay on 4, 14, and 50kPa collagen-coated polyacrylamide gels. Total cell migration distance was measured over time, as were nuclear alignment and nuclear:total β-catenin as measures of cell directedness and cell-cell junction integrity, respectively. In addition, fibronectin fibers were examined as a measure of extracellular matrix deposition and remodeling. We now show that endothelial cells collectively migrate farther on stiffer substrates by 24h. Cells were more directed in the migration direction on intermediate stiffness substrates from 12 to 24h, with an alignment peak 400-700µm back from the migratory interface. However, cells on the softest substrates had the highest cell-cell junction integrity. Cells on all substrates deposited fibronectin, however fibronectin fibers were most linear and aligned on the stiffer substrates. When Rho kinase (ROCK) was inhibited with Y27632, cells on soft substrates migrated farther and cells on both soft and stiff substrates were more directed. When α5 integrin was knocked down with siRNA, cells on stiffer substrates did not migrate as far and were less directed. These data suggest that ROCK-mediated myosin contractility inhibits endothelial cell collective migration on soft substrates, while cell-matrix interactions are critical to endothelial cell collective migration on stiff substrates.

  3. P-cadherin promotes collective cell migration via a Cdc42-mediated increase in mechanical forces

    Science.gov (United States)

    Plutoni, Cédric; Bazellieres, Elsa; Le Borgne-Rochet, Maïlys; Comunale, Franck; Brugues, Agusti; Séveno, Martial; Planchon, Damien; Thuault, Sylvie; Morin, Nathalie; Bodin, Stéphane; Trepat, Xavier

    2016-01-01

    Collective cell migration (CCM) is essential for organism development, wound healing, and metastatic transition, the primary cause of cancer-related death, and it involves cell–cell adhesion molecules of the cadherin family. Increased P-cadherin expression levels are correlated with tumor aggressiveness in carcinoma and aggressive sarcoma; however, how P-cadherin promotes tumor malignancy remains unknown. Here, using integrated cell biology and biophysical approaches, we determined that P-cadherin specifically induces polarization and CCM through an increase in the strength and anisotropy of mechanical forces. We show that this mechanical regulation is mediated by the P-cadherin/β-PIX/Cdc42 axis; P-cadherin specifically activates Cdc42 through β-PIX, which is specifically recruited at cell–cell contacts upon CCM. This mechanism of cell polarization and migration is absent in cells expressing E- or R-cadherin. Thus, we identify a specific role of P-cadherin through β-PIX–mediated Cdc42 activation in the regulation of cell polarity and force anisotropy that drives CCM. PMID:26783302

  4. Autologous peripheral blood stem cell harvest: Collection efficiency and factors affecting it

    Directory of Open Access Journals (Sweden)

    Aseem K Tiwari

    2016-01-01

    Full Text Available Background: Harvest of hematopoietic progenitor cells via leukapheresis is being used increasingly for transplants in India. Adequate yield of cells per kilogram body weight of recipient is required for successful engraftment. Collection efficiency (CE is an objective quality parameter used to assess the quality of leukapheresis program. In this study, we calculated the CE of the ComTec cell separator (Fresenius Kabi, Germany using two different formulae (CE1 and CE2 and analyzed various patient and procedural factors, which may affect it. Materials and Methods: One hundred and one consecutive procedures in 77 autologous donors carried out over 3 years period were retrospectively reviewed. Various characteristics like gender, age, weight, disease status, hematocrit, preprocedure total leukocyte count, preprocedure CD34 positive (CD34+ cells count, preprocedure absolute CD34+ cell count and processed apheresis volume effect on CE were compared. CE for each procedure was calculated using two different formulae, and results were compared using statistical correlation and regression analysis. Results: The mean CE1 and CE2 was 41.2 and 49.1, respectively. CE2 appeared to be more accurate indicator of overall CE as it considered the impact of continued mobilization of stem cells during apheresis procedure, itself. Of all the factors affecting CE, preprocedure absolute CD34+ was the only independent factor affecting CE. Conclusion: The only factor affecting CE was preprocedure absolute CD34+ cells. Though the mean CE2 was higher than CE1, it was not statistically significant.

  5. Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule.

    Science.gov (United States)

    Christensen, E I; Birn, H

    2001-04-01

    The multiligand, endocytic receptors megalin and cubilin are colocalized in the renal proximal tubule. They are heavily expressed in the apical endocytic apparatus. Megalin is a 600-kDa transmembrane protein belonging to the low-density lipoprotein-receptor family. The cytoplasmic tail contains three NPXY motifs that mediate the clustering in coated pits and are possibly involved in signaling functions. Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is a 460-kDa receptor with no transmembrane domain and no known signal for endocytosis. Because the two receptors bind each other with high affinity and colocalize in several tissues, it is highly conceivable that megalin mediates internalization of cubilin and its ligands. Both receptors are important for normal tubular reabsorption of proteins, including albumin. Among the proteins normally filtered in the glomeruli, cubilin has been shown to bind albumin, immunoglobulin light chains, and apolipoprotein A-I. The variety of filtered ligands identified for megalin include vitamin-binding proteins, hormones, enzymes, apolipoprotein H, albumin, and beta(2)- and alpha(1)-microglobulin. Loss of these proteins and vitamins in the urine of megalin-deficient mice illustrates the physiological importance of this receptor.

  6. Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions.

    Science.gov (United States)

    Amora, Daniela N; Costa Martins, Alice M; Roeser, Nancy; Senter, Ruth; Ostrowsky, Tiffany; Weinberg, Joel M; Monteiro, Helena S A

    2008-12-15

    In the present study we show that phospholipases A2 isolated from porcine pancreas (PP-PLA2) and Crotalus durissus terrificus snake venom (SV-PLA2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA(2)s induced increases in non-esterified fatty acid (NEFA) levels (microg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA2, and were dose-dependent for the crotalic PLA2 group. Furthermore, snake venom PLA2 induced a decrease in mitochondrial membrane potential (DeltaPsi(m)) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on DeltaPsi(m) measured with safranin O. Incubation with pancreatic and crotalic phospholipases A2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.

  7. T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Janine Hallerdei

    Full Text Available We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

  8. Objectives, Outlines, and Preparation for the Resist Tubule Space Experiment to Understand the Mechanism of Gravity Resistance in Plants

    Science.gov (United States)

    Hoson, Takayuki; Akamatsu, Haruhiko; Soga, Kouichi; Wakabayashi, Kazuyuki; Hashimoto, Hirofumi; Yamashita, Masamichi; Hasegawa, Katsuya; Yano, Sachiko; Omori, Katsunori; Ishioka, Noriaki; Matsumoto, Shohei; Kasahara, Haruo; Shimazu, Toru; A. Baba, Shoji; Hashimoto, Takashi

    Gravity resistance is a principal graviresponse in plants. In resistance to hypergravity, the gravity signal may be perceived by the mechanoreceptors located on the plasma membrane, and then transformed and transduced via the structural continuum or physiological continuity of cortical microtubules-plasma membrane-cell wall, leading to an increase in the cell wall rigidity as the final response. The Resist Tubule experiment, which will be conducted in the Kibo Module on the International Space Station, aims to confirm that this hypothesis is applicable to resistance to 1 G gravity. There are two major objectives in the Resist Tubule experiment. One is to quantify the contributions of cortical microtubules to gravity resistance using Arabidopsis tubulin mutants with different degrees of defects. Another objective is to analyze the modifications to dynamics of cortical microtubules and membrane rafts under microgravity conditions on-site by observing green fluorescent protein (GFP)-expressing Arabidopsis lines with the fluorescence microscope in the Kibo. We have selected suitable mutants, developed necessary hardware, and fixed operation procedure for the experiment.

  9. 78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

    Science.gov (United States)

    2013-02-28

    ... Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research SUMMARY: In compliance with... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be... within 60 days of the date of this publication. Proposed Collection: Request for Human Embryonic Stem...

  10. 77 FR 2734 - Proposed Collection; Comment Request: Solar Cell: A Mobile UV Manager for Smart Phones (NCI)

    Science.gov (United States)

    2012-01-19

    ... HUMAN SERVICES National Institutes of Health Proposed Collection; Comment Request: Solar Cell: A Mobile... (OMB) for review and approval. Proposed Collection: Title: Solar Cell: A Mobile UV Manager for Smart... technology to aid users in protecting their skin from damaging ultraviolet radiation (UV) in sunlight,...

  11. 77 FR 4334 - Proposed Collection; Comment Request; Solar Cell: A Mobile UV Manager for Smart Phones (NCI)

    Science.gov (United States)

    2012-01-27

    ... HUMAN SERVICES National Institutes of Health Proposed Collection; Comment Request; Solar Cell: A Mobile... (OMB) for review and approval. Proposed Collection: Title: Solar Cell: A Mobile UV Manager for Smart... technology to aid users in protecting their skin from damaging ultraviolet radiation (UV) in sunlight,...

  12. Perfusion Method for Intra-bone Marrow Collection and Stem Cell Transplantation: A Critical Review.

    Science.gov (United States)

    Korrapati, Narasimhulu; Nanganuru, Harikrishna Yadav

    2014-03-19

    A bone marrow transplant is a procedure to replace damaged or destroyed bone marrow with healthy bone marrow stem cells. Bone marrow is the soft, fatty tissue inside our bones. Bone marrow transplantation (BMT) is a powerful strategy for the treatment of leukemia, aplastic anemia, congenital immunodeficiency and autoimmune diseases. In humans, bone marrow cells (BMCs) have usually been collected by multiple bone marrow aspirations from the iliac crest. We have established a new "perfusion" method for collecting BMCs with minimal contamination with the peripheral blood using the long bones of cynomolgus monkeys. This method has proven to be a simple and safe method for harvesting BMCs and reduces the risk of acute graft versus host disease in allogeneic BMT. Intra-bone marrow-BMT (IBM-BMT) provides distinct advantages because it recruits donor-derived hematopoietic stem cells and mesenchymal stem cells. IBM-BMT has been shown to currently be the best strategy for allogeneic BMT. Here we review the perfusion method (for harvesting BMCs) and IBM-BMT (for their transplantation) and show that this combination will become a powerful new clinical strategy for allogeneic BMT.

  13. Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

    Directory of Open Access Journals (Sweden)

    Kui-Jin Kim

    Full Text Available Basal-like breast carcinomas (BLCs present with extratumoral lymphovascular invasion, are highly metastatic, presumably through a hematogenous route, have augmented expression of CD44 oncoprotein and relatively low levels of retinoblastoma (Rb tumor suppressor. However, the causal relation among these features is not clear. Here, we show that Rb acts as a key suppressor of multiple stages of metastatic progression. Firstly, Rb suppresses collective cell migration (CCM and CD44-dependent formation of F-actin positive protrusions in vitro and cell-cluster based lymphovascular invasion in vivo. Secondly, Rb inhibits the release of single cancer cells and cell clusters into the hematogenous circulation and subsequent metastatic growth in lungs. Finally, CD44 expression is required for collective motility and all subsequent stages of metastatic progression initiated by loss of Rb function. Altogether, our results suggest that Rb/CD44 pathway is a crucial regulator of CCM and metastatic progression of BLCs and a promising target for anti-BLCs therapy.

  14. Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease.

    Science.gov (United States)

    Fesenko, Irina; Franklin, Danielle; Garnett, Paul; Bass, Paul; Campbell, Sara; Hardyman, Michelle; Wilson, David; Hanley, Neil; Collins, Jane

    2010-10-01

    The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm-Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.

  15. Spatial moment dynamics for collective cell movement incorporating a neighbour-dependent directional bias.

    Science.gov (United States)

    Binny, Rachelle N; Plank, Michael J; James, Alex

    2015-05-06

    The ability of cells to undergo collective movement plays a fundamental role in tissue repair, development and cancer. Interactions occurring at the level of individual cells may lead to the development of spatial structure which will affect the dynamics of migrating cells at a population level. Models that try to predict population-level behaviour often take a mean-field approach, which assumes that individuals interact with one another in proportion to their average density and ignores the presence of any small-scale spatial structure. In this work, we develop a lattice-free individual-based model (IBM) that uses random walk theory to model the stochastic interactions occurring at the scale of individual migrating cells. We incorporate a mechanism for local directional bias such that an individual's direction of movement is dependent on the degree of cell crowding in its neighbourhood. As an alternative to the mean-field approach, we also employ spatial moment theory to develop a population-level model which accounts for spatial structure and predicts how these individual-level interactions propagate to the scale of the whole population. The IBM is used to derive an equation for dynamics of the second spatial moment (the average density of pairs of cells) which incorporates the neighbour-dependent directional bias, and we solve this numerically for a spatially homogeneous case.

  16. Classification of solar cells according to mechanisms of charge separation and charge collection.

    Science.gov (United States)

    Kirchartz, Thomas; Bisquert, Juan; Mora-Sero, Ivan; Garcia-Belmonte, Germà

    2015-02-14

    In the last decade, photovoltaics (PV) has experienced an important transformation. Traditional solar cells formed by compact semiconductor layers have been joined by new kinds of cells that are constituted by a complex mixture of organic, inorganic and solid or liquid electrolyte materials, and rely on charge separation at the nanoscale. Recently, metal organic halide perovskites have appeared in the photovoltaic landscape showing large conversion efficiencies, and they may share characteristics of the two former types. In this paper we provide a general description of the photovoltaic mechanisms of the single absorber solar cell types, combining all-inorganic, hybrid and organic cells into a single framework. The operation of the solar cell relies on a number of internal processes that exploit internal charge separation and overall charge collection minimizing recombination. There are two main effects to achieve the required efficiency, first to exploit kinetics at interfaces, favouring the required forward process, and second to take advantage of internal electrical fields caused by a built-in voltage and by the distribution of photogenerated charges. These principles represented by selective contacts, interfaces and the main energy diagram, form a solid base for the discussion of the operation of future types of solar cells. Additional effects based on ferroelectric polarization and ionic drift provide interesting prospects for investigating new PV effects mainly in the perovskite materials.

  17. Regulatory pathways for ATP-binding cassette transport proteins in kidney proximal tubules

    NARCIS (Netherlands)

    Masereeuw, R.; Russel, F.G.M.

    2012-01-01

    The ATP-binding cassette transport proteins (ABC transporters) represent important determinants of drug excretion. Protective or excretory tissues where these transporters mediate substrate efflux include the kidney proximal tubule. Regulation of the transport proteins in this tissue requires elabor

  18. Effect of depth and tubule direction on ultimate tensile strength of human coronal dentin.

    Science.gov (United States)

    Inoue, Satoshi; Pereira, Patricia N R; Kawamoto, Chiharu; Nakajima, Masatoshi; Koshiro, Kenichi; Tagami, Junji; Carvalho, Ricardo M; Pashley, David H; Sano, Hidehiko

    2003-03-01

    The purpose of this study was to evaluate the effect of dentin depth and tubule direction on the ultimate tensile strength (UTS) of human dentin. Dentin slabs of 0.5-mm thickness were trimmed either from the mesial and distal (for specimens with the tubules parallel to the tensile force; parallel group) or from the occlusal and pulpal surfaces (perpendicular group) to reduce the cross-sectional area of the superficial, middle, and deep regions to 0.25 mm2, and subjected to microtensile testing. From SEM photomicrographs of the fractured specimens of the parallel group, the tubule density was investigated. For both parallel and perpendicular groups, superficial dentin showed a significantly higher UTS than deep dentin. The tubule density of superficial dentin was significantly lower than that of middle and deep dentin. When performing the microtensile bond test to deep dentin, it is possible that cohesive failure of dentin can occur at relatively low tensile stresses.

  19. Purine transport by malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster.

    Science.gov (United States)

    Sullivan, D T; Bell, L A; Paton, D R; Sullivan, M C

    1979-06-01

    Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (pp) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.

  20. Resin Adaptation of Radicular Dentin Tubules after Endodontic Instrumentation and Acid Etching.

    Science.gov (United States)

    1983-02-01

    intracanal irrigant . After completion of the Carson L. Mader 15 -1 endodontic instrumentation, the canals were flushed with 25 ml. of sterile saline...I RD-Ai26 872 RESIN ADAPTATION OF RADICULAR DENTIN TUBULES AFTER / I ENDODONTIC INSTRUMENTATION AND ACID ETCHING(U) WALTER I REED ARMY INST OF...Adaptation to Radicular Dentin Tubules SbisoofpeAfter Endodontic Instrumentation and Acid Etching 1982-1983 6. PERFORMING ORG. REPORTNUMBER -, AUTHOR(a) S

  1. Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish.

    Science.gov (United States)

    Kitatani, Ryuju; Yamada, Mayu; Kamio, Michiya; Nagai, Hiroshi

    2015-01-01

    A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation.

  2. Effects of ammonium hexafluorosilicate concentration on dentin tubule occlusion and composition of the precipitate.

    Science.gov (United States)

    Suge, Toshiyuki; Kawasaki, Akiko; Ishikawa, Kunio; Matsuo, Takashi; Ebisu, Shigeyuki

    2010-01-01

    Ammonium hexafluorosilicate [SiF: (NH(4))(2)SiF(6)] was prepared in order to overcome the tooth discoloration caused by diamine silver fluoride [AgF: (NH(3))(2)AgF] application. We employed a single concentration of SiF solution in our previous study; therefore, it is still unclear how the concentration of SiF solution affects the occlusion of dentin tubules and composition of the precipitate. The aim of this study was to evaluate the effects of changing the concentration of SiF on its clinical use as a dentin hypersensitivity treatment. To simulate dentin tubules subject to dentin hypersensitivity, dentin disks were treated with EDTA for 2 min. Then, the disks were treated with several concentrations of SiF solution (from 100 to 19,400 ppm) for 3 min. The occlusion of dentin tubules was evaluated using scanning electron microscopy (SEM), and the composition of the precipitate formed in the tubules after SiF treatment was assessed using energy dispersive X-ray analysis (EDXA). SEM photographs demonstrated that dentin tubules after treatment with SiF were occluded homogeneously and fully regardless of the concentration of SiF solution. The Ca/P molar ratio of the precipitate formed in dentin tubules after SiF treatment was increased with the concentration of SiF solution. It was concluded that the capacity to occlude dentin tubules was the same regardless of the concentration of SiF solution. However, the composition of the precipitate formed in the tubules was dependent on the concentration of SiF solution.

  3. Renal proximal tubule Na,K-ATPase is controlled by CREB-regulated transcriptional coactivators as well as salt-inducible kinase 1.

    Science.gov (United States)

    Taub, Mary; Garimella, Sudha; Kim, Dongwook; Rajkhowa, Trivikram; Cutuli, Facundo

    2015-12-01

    Sodium reabsorption by the kidney is regulated by locally produced natriuretic and anti-natriuretic factors, including dopamine and norepinephrine, respectively. Previous studies indicated that signaling events initiated by these natriuretic and anti-natriuretic factors achieve their effects by altering the phosphorylation of Na,K-ATPase in the renal proximal tubule, and that protein kinase A (PKA) and calcium-mediated signaling pathways are involved. The same signaling pathways also control the transcription of the Na,K-ATPase β subunit gene atp1b1 in renal proximal tubule cells. In this report, evidence is presented that (1) both the recently discovered cAMP-regulated transcriptional coactivators (CRTCs) and salt-inducible kinase 1 (SIK1) contribute to the transcriptional regulation of atp1b1 in renal proximal tubule (RPT) cells and (2) renal effectors, including norepinephrine, dopamine, prostaglandins, and sodium, play a role. Exogenously expressed CRTCs stimulate atp1b1 transcription. Evidence for a role of endogenous CRTCs includes the loss of transcriptional regulation of atp1b1 by a dominant-negative CRTC, as well as by a CREB mutant, with an altered CRTC binding site. In a number of experimental systems, SIK phosphorylates CRTCs, which are then sequestered in the cytoplasm, preventing their nuclear effects. Consistent with such a role of SIK in primary RPT cells, atp1b1 transcription increased in the presence of a dominant-negative SIK1, and in addition, regulation by dopamine, norepinephrine, and monensin was disrupted by a dominant-negative SIK1. These latter observations can be explained if SIK1 is phosphorylated and inactivated in the presence of these renal effectors. Our results support the hypothesis that Na,K-ATPase in the renal proximal tubule is regulated at the transcriptional level via SIK1 and CRTCs by renal effectors, in addition to the previously reported control of the phosphorylation of Na,K-ATPase.

  4. Investigation of dentinal tubule occlusion using FIB-SEM milling and EDX.

    Science.gov (United States)

    Earl, J S; Ward, M B; Langford, R M

    2010-01-01

    The aim of this study was to evaluate in vitro the dentin tubule occluding effect of an 8% strontium acetate dentifrice (Sensodyne Rapid Relief) compared to patent dentin tubules using modern sample preparation, imaging, and analysis techniques. Etched dentin discs, either untreated or treated with the dentifrice, were analyzed by preparing cross-sections using focused ion beam scanning electron microscopy (FIB-SEM) milling, and the strontium presence mapped using energy dispersive X-ray spectroscopy (EDX). Surface imaging showed the dentifrice had coated the treated sample. Sub-surface information gained by preparing longitudinal cross-sections of the treated samples showed the tubule openings to be plugged, and EDX mapping of the cross-section confirmed enhanced strontium levels within the tubules several microns below the treatment surface. The combination of modern sample preparation, imaging, and analysis techniques employed in this study has shown that the 8% strontium acetate dentifrice occludes dentin tubules. EDX analysis has shown the presence of strontium within the dentin tubules, with elemental maps illustrating how the strontium has been incorporated into the dentin.

  5. EHD1 mediates vesicle trafficking required for normal muscle growth and tubule development

    Science.gov (United States)

    Posey, Avery D.; Swanson, Kaitlin E.; Alvarez, Manuel G.; Krishnan, Swathi; Earley, Judy E.; Band, Hamid; Pytel, Peter; McNally, Elizabeth M.; Demonbreun, Alexis R.

    2014-01-01

    EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton. PMID:24440153

  6. EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.

    Science.gov (United States)

    Posey, Avery D; Swanson, Kaitlin E; Alvarez, Manuel G; Krishnan, Swathi; Earley, Judy U; Band, Hamid; Pytel, Peter; McNally, Elizabeth M; Demonbreun, Alexis R

    2014-03-15

    EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Enhancing electron collection efficiency and effective diffusion length in dye-sensitized solar cells.

    Science.gov (United States)

    Wong, Daniel Kwan-Pang; Ku, Chen-Hao; Chen, Yen-Ru; Chen, Guan-Ren; Wu, Jih-Jen

    2009-10-19

    Intensity-modulated photocurrent spectroscopy and intensity-modulated photovoltage spectroscopy are employed to measure the dynamics of electron transport and recombination in the ZnO nanowire (NW) array-ZnO/layered basic zinc acetate (LBZA) nanoparticle (NP) composite dye-sensitized solar cells (DSSCs). The roles of the vertical ZnO NWs and insulating LBZA in the electron collection and transport in DSSCs are investigated by comparing the results to those in the TiO(2)-NP, horizontal TiO(2)-NW and vertical ZnO-NW-array DSSCs. The electron transport rate and electron lifetime in the ZnO NW/NP composite DSSC are superior to those in the conventional TiO(2)-NP cell due to the existence of the vertical ZnO NWs and insulating LBZA. It indicates that the ZnO NW/NP composite anode is able to sustain efficient electron collection over much greater thickness than the TiO(2)-NP cell does. Consequently, a larger effective electron diffusion length is available in the ZnO composite DSSC.

  8. Treatment of dentinal tubules by Nd:YAG laser

    Science.gov (United States)

    Chmelíčkova, Hana; Zapletalova, Zdeňka; Peřina, Jan, Jr.; Novotný, Radko; Kubínek, Roman; Stranyánek, Martin

    2005-08-01

    Symptom of cervical dentine hypersensitivity attacks from 10% to 15% of population and causes an uncomfortable pain during contact with any matter. Sealing of open dentinal tubules is one of the methods to reach insensibility. Laser as a source of coherent radiation is used to melt dentine surface layers. Melted dentine turns to hard mass with a smooth, non-porous surface. Simulation of this therapy was made in vitro by means of LASAG Nd:YAG pulsed laser system KLS 246-102. Eighty human extracted teeth were cut horizontally to obtain samples from 2 mm to 3 mm thick. First experiments were done on cross section surfaces to find an optimal range of laser parameters. A wide range of energies from 30 mJ to 210 mJ embedded in 0,3 ms long pulse was tested. Motion in X and Y axes was ensured by a CNC driven table and the pulse frequency 15 Hz was chosen to have a suitable overlap of laser spots. Some color agents were examined with the aim to improve surface absorption. Scanning Electron Microscopy was used to evaluate all samples and provided optimal values of energies around 50 J.cm-2. Next experiments were done with the beam oriented perpendicularly to a root surface, close to the real situation. Optical fibers with the diameter of 0,6 mm and 0,2 mm were used to guide a laser beam to teeth surfaces. Laser processing heads with lens F = 100 mm and F = 50 mm were used. The best samples were investigated by means of the Atomic Force Microscopy.

  9. Human Urine as a Noninvasive Source of Kidney Cells

    Directory of Open Access Journals (Sweden)

    Fanny Oliveira Arcolino

    2015-01-01

    Full Text Available Urine represents an unlimited source of patient-specific kidney cells that can be harvested noninvasively. Urine derived podocytes and proximal tubule cells have been used to study disease mechanisms and to screen for novel drug therapies in a variety of human kidney disorders. The urinary kidney stem/progenitor cells and extracellular vesicles, instead, might be promising for therapeutic treatments of kidney injury. The greatest advantages of urine as a source of viable cells are the easy collection and less complicated ethical issues. However, extensive characterization and in vivo studies still have to be performed before the clinical use of urine-derived kidney progenitors.

  10. Highly Conformal Ni Micromesh as a Current Collecting Front Electrode for Reduced Cost Si Solar Cell.

    Science.gov (United States)

    Gupta, Nikita; Rao, K D Mallikharjuna; Gupta, Ritu; Krebs, Frederik C; Kulkarni, Giridhar U

    2017-02-17

    Despite relatively high manufacturing cost, crystalline-Si solar cell continues to hold promising future due to its high energy conversion efficiency and long life. As regards cost, one pertinent issue is the top electrode metallization of textured cell surface, which typically involves screen printing of silver paste. The associated disadvantages call for alternative methods that can lower the cost without compromising the solar cell efficiency. In the present work, a highly interconnected one dimensional (1D) metal wire network has been employed as front electrode on conventional Si wafers. Here for the first time, we report an innovative solution based crackle templating method for conformal metal wire network patterning over large textured surfaces. Laser beam induced current mapping showed uniform photocurrent collection by the electrodes without any shadow losses. With electroless deposition of Ni wire network on corrugated solar cell, a short circuit current of 33.28 mA/cm2 was obtained in comparison to 20.53 mA/cm2 without the network electrode. On comparing the efficiency with the conventional cells with screen printed electrodes, a 20% increment in efficiency has been observed. Importantly, the estimated manufacturing cost is three orders lower.

  11. Treatment Options for Renal Cell Cancer

    Science.gov (United States)

    ... cell cancer is a disease in which malignant (cancer) cells form in tubules of the kidney. Renal cell ... diagnosed, tests are done to find out if cancer cells have spread within the kidney or to other ...

  12. General Information about Renal Cell Cancer

    Science.gov (United States)

    ... cell cancer is a disease in which malignant (cancer) cells form in tubules of the kidney. Renal cell ... diagnosed, tests are done to find out if cancer cells have spread within the kidney or to other ...

  13. Treatment Option Overview (Renal Cell Cancer)

    Science.gov (United States)

    ... cell cancer is a disease in which malignant (cancer) cells form in tubules of the kidney. Renal cell ... diagnosed, tests are done to find out if cancer cells have spread within the kidney or to other ...

  14. Sterile subperiosteal fluid collections accompanying orbital wall infarction in sickle-cell disease.

    Science.gov (United States)

    Huckfeldt, Rachel M; Shah, Ankoor S

    2014-10-01

    Infarction of the orbital wall is an uncommon manifestation of sickle cell disease (SCD) that may mimic an infectious process. We report a patient with two separate orbital infarctions with different presenting symptoms involving different bones. Radiologic-guided sampling of a periosteal fluid collection in the first episode showed likely sterile inflammatory exudates. This case highlights the range of findings in orbital wall infarction in SCD as well as helpful clinical and imaging entities that may differentiate infarction from infection, allowing early diagnosis and appropriate management.

  15. Minimization of the effect of the collecting grid in a solar cell based silicon

    Energy Technology Data Exchange (ETDEWEB)

    Cheknane, A.; Benyoucef, B. [Laboratoire des Materiaux et Energies Renouvelables, Tlemcen (Algeria); Charles, J.-P. [MOPS, SUPELEC, Metz (France); Zerdoum, R. [Riyadh College of Technology, Riyadh (Saudi Arabia); Trari, M. [Laboratoire de Stockage et de Valorisation des Energies Renouvelables, Alger (Algeria)

    2005-05-01

    The solar cells collecting grids present a serious problem and more particularly under solar concentration. Our contribution in this article is to seek the best compromise between shadow effect and series resistance effect. The cell considered here is of Si (silicon) type, n{sup +}p with circular geometry (radius {alpha} = 4.9cm), a silver metallization ({rho}M = 1.6 x 10{sup -6} {omega}cm), and a contact resistivity of {rho}C = 10{sup -5} {omega}cm. Our calculations are made under the condition of AM1.5 with 1 sun concentration. The various power losses caused by this grid are: losses due to the grid shadow, losses in grain boundaries due to the metal/semiconductor contact, power dissipated in the resistance of layer between bars, and losses in the grid metallization. (author)

  16. Midline-derived Shh regulates mesonephric tubule formation through the paraxial mesoderm.

    Science.gov (United States)

    Murashima, Aki; Akita, Hiroki; Okazawa, Mika; Kishigami, Satoshi; Nakagata, Naomi; Nishinakamura, Ryuichi; Yamada, Gen

    2014-02-01

    During organogenesis, Sonic hedgehog (Shh) possesses dual functions: Shh emanating from midline structures regulates the positioning of bilateral structures at early stages, whereas organ-specific Shh locally regulates organ morphogenesis at later stages. The mesonephros is a transient embryonic kidney in amniote, whereas it becomes definitive adult kidney in some anamniotes. Thus, elucidating the regulation of mesonephros formation has important implications for our understanding of kidney development and evolution. In Shh knockout (KO) mutant mice, the mesonephros was displaced towards the midline and ectopic mesonephric tubules (MTs) were present in the caudal mesonephros. Mesonephros-specific ablation of Shh in Hoxb7-Cre;Shh(flox/-) and Sall1(CreERT2/+);Shh(flox/-) mice embryos indicated that Shh expressed in the mesonephros was not required for either the development of the mesonephros or the differentiation of the male reproductive tract. Moreover, stage-specific ablation of Shh in Shh(CreERT2/flox) mice showed that notochord- and/or floor plate-derived Shh were essential for the regulation of the number and position of MTs. Lineage analysis of hedgehog (Hh)-responsive cells, and analysis of gene expression in Shh KO embryos suggested that Shh regulated nephrogenic gene expression indirectly, possibly through effects on the paraxial mesoderm. These data demonstrate the essential role of midline-derived Shh in local tissue morphogenesis and differentiation.

  17. Grain engineering: How nanoscale inhomogeneities can control charge collection in solar cells

    Energy Technology Data Exchange (ETDEWEB)

    West, Bradley M.; Stuckelberger, Michael; Guthrey, Harvey; Chen, Lei; Lai, Barry; Maser, Jörg; Rose, Volker; Shafarman, William; Al-Jassim, Mowafak; Bertoni, Mariana I.

    2017-02-01

    Statistical and correlative analysis are increasingly important in the design and study of new materials, from semiconductors to metals. Non-destructive measurement techniques, with high spatial resolution, capable of correlating composition and/or structure with device properties, are few and far between. For the case of polycrystalline and inhomogeneous materials, the added challenge is that nanoscale resolution is in general not compatible with the large sampling areas necessary to have a statistical representation of the specimen under study. For the study of grain cores and grain boundaries in polycrystalline solar absorbers this is of particular importance since their dissimilar behavior and variability throughout the samples makes it difficult to draw conclusions and ultimately optimize the material. In this study, we present a nanoscale in-operando approach based on the multimodal utilization of synchrotron nano x-ray fluorescence and x-ray beam induced current collected for grain core and grain boundary areas and correlated pixel-by-pixel in fully operational Cu(In(1-x)Gax)Se2Cu(In(1-x)Gax)Se2 solar cells. We observe that low gallium cells have grain boundaries that over perform compared to the grain cores and high gallium cells have boundaries that under perform. These results demonstrate how nanoscale correlative X-ray microscopy can guide research pathways towards grain engineering low cost, high efficiency solar cells.

  18. The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme.

    Science.gov (United States)

    Alford, Lea M; Stoddard, Daniel; Li, Jennifer H; Hunter, Emily L; Tritschler, Douglas; Bower, Raqual; Nicastro, Daniela; Porter, Mary E; Sale, Winfield S

    2016-06-01

    We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.

  19. How complex elements can form cell membrane and modular network collectively

    CERN Document Server

    Tanaka, D

    2006-01-01

    One degree of freedom is sufficient for intra-elements. In Japan, a huge project studying soft matter involves a budget of the order of a billion yen. This is just one aspect showing the world-wide interest in non-equilibrium elements whose internal dynamics interacts with macroscopic or mesoscopic order of elements. The elements in this paper denote symptoms such as a bacterium having an internal network of genes and proteins, a reactive droplet in reaction-diffusion systems, a neuron in networks, etc. These elements exhibit not only spatio-temporal patterns but also collective functions. For instance, the cohort migration of mammalian cells forms tissue patterns, and the Proteus mirabilis effectively invades human urothelial cells by swarming. Further, swarm intelligence has been extensively studied in order to enable a collection of simple robots to perform advanced tasks. Here, we show a simple model derived by means of mathematical techniques to study the cross-cutting phenomenon underlying the above sys...

  20. Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

    Science.gov (United States)

    Schenk, Laura K; Bolger, Steven J; Luginbuhl, Kelli; Gonzales, Patricia A; Rinschen, Markus M; Yu, Ming-Jiun; Hoffert, Jason D; Pisitkun, Trairak; Knepper, Mark A

    2012-06-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

  1. Study of serum ferritin in donors of two red blood cells units collected by apheresis.

    Science.gov (United States)

    González, Maria Luz Dobao; Maia, Salome; Mesquita, Paula; Bessa, Milena

    2013-10-01

    To analyze the recovery of iron stores without supplementation, when keeping an interval of six months between donations. From April 2007 to May 2011, 308 regular and voluntary donors were selected. The apheresis collections were performed using ALYX® Component Collection System-Fenwal™. The hematological parameters were analyzed using the Cell DIN Sapphire - Abbot Diagnostics, and the serum ferritin by sandwich immunoassay method with fluorescence detection in final phase (ELFA) - Vidas® Ferritin-Biomérieux SA. A descriptive statistical analysis was performed for each hematological parameters and serum ferritin. The median hemoglobin concentration was 15.6g/dL (14, 18.4) in the first procedure and remains constant at subsequent donations. The ferritin median concentration was 64.6 μg/L (7.2, 886). A decrease of 15.6% was observed when compared the first to the second procedure with a median 54.6 μg/L (8.3, 213.7). Paradoxically, this decrease is not evident in the subsequent procedures, where an increase of 14.6% and 3.4% for the third and fourth procedure respectively was observed. Changes in ferritin values show statistically significant differences between the first and second collection, but this difference disappeared in subsequent donations. The analysis of MCH in each collection indicates that the significant difference between first and second donation (p1-2ferritin found between procedures and the beginning of the stabilization of ferritin levels. The determination of ferritin appears not to be the most important parameter to consider at the time of donor selection and suggests that other factors unrelated to the donation may play a significant role. A decrease in serum ferritin was observed at the beginning, but it seems to attend a recovery and stabilization in the successive procedures. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Feasibility of collecting buccal cell DNA by mail in a cohort study.

    Science.gov (United States)

    Le Marchand, L; Lum-Jones, A; Saltzman, B; Visaya, V; Nomura, A M; Kolonel, L N

    2001-06-01

    This study assessed the feasibility of obtaining buccal cell DNA by mail from participants in a large, community-based cohort study in Hawaii. Mouthwash collection kits were sent to a total of 355 randomly selected Japanese, Caucasian, and Hawaiian cohort members. Subjects were requested to swish 10 ml of mouthwash in their mouth for 60 s and expel it into a collection cup, which they mailed back to our laboratory. Half of the subjects were requested to collect a second sample. After up to two mailings and two reminder phone calls, two-thirds of the subjects returned a sample. The participation rate was lower for Hawaiians (59.0%) than for Caucasians (68.1%) and Japanese (76.3%). Participation was not affected by requesting two specimens. Participants did not differ from the total sample in terms of education and smoking status. The mean DNA yield was lower in females (41.7 microg) than males (53.4 microg) and in Japanese (37.8 microg) as compared with Hawaiians (51.9 microg) and Caucasians (54.5 microg). For subjects who returned two samples, the DNA yields were similar when both specimens were extracted in the same batch. All samples were successfully genotyped for polymorphisms in the CYP1A1, CYP2E1, GSTM1, GSTT1, and NQO1 genes by PCR-RFLP. From these and previous data, we conclude that, in situations where blood samples cannot be obtained, mail collection of mouthwash samples should be considered because it yields substantial amounts of high-quality genomic DNA for large numbers of study subjects.

  3. Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse

    Directory of Open Access Journals (Sweden)

    Segall Jeffrey E

    2003-08-01

    Full Text Available Abstract Background cDNA microarrays have the potential to identify the genes involved in invasion and metastasis. However, when used with whole tumor tissue, the results average the expression patterns of different cell types. We have combined chemotaxis-based cell collection of the invasive subpopulation of cells within the primary tumor with array-based gene expression analysis to identify the genes necessary for the process of carcinoma cell invasion. Results Invasive cells were collected from live primary tumors using microneedles containing chemotactic growth factors to mimic chemotactic signals thought to be present in the primary tumor. When used with mammary tumors of rats and mice, carcinoma cells and macrophages constitute the invasive cell population. Microbeads conjugated with monoclonal anti-CD11b (Mac-1α antibodies were used to separate macrophages from carcinoma cells. We utilized PCR-based cDNA amplification from small number of cells and compared it to the quality and complexity of conventionally generated cDNA to determine if amplified cDNA could be used with fidelity for array analysis of this cell population. These techniques showed a very high level of correlation indicating that the PCR based amplification technique yields a cDNA population that resembles, with high fidelity, the original template population present in the small number of cells used to prepare the cDNA for use with the chip. Conclusions The specific collection of invasive cells from a primary tumor and the analysis of gene expression in these cells are is now possible. By further comparing the gene expression patterns of cells collected by invasion into microneedles with that of carcinoma cells obtained from the whole primary tumor, the blood, and whole metastatic tumors, genes that contribute to the invasive process in carcinoma cells may be identified.

  4. Titanium oxide morphology controls charge collection efficiency in quantum dot solar cells.

    Science.gov (United States)

    Kolay, Ankita; Kumar, P Naresh; Kumar, Sarode Krishna; Deepa, Melepurath

    2017-02-08

    Charge transfer at the TiO2/quantum dots (QDs) interface, charge collection at the TiO2/QDs/current collector (FTO or SnO2:F) interface, and back electron transfer at the TiO2/QDs/S(2-) interface are processes controlled by the electron transport layer or TiO2. These key processes control the power conversion efficiencies (PCEs) of quantum dot solar cells (QDSCs). Here, four TiO2 morphologies, porous nanoparticles (PNPs), nanowires (NWs), nanosheets (NSHs) and nanoparticles (NPs), were sensitized with CdS and the photovoltaic performances were compared. The marked differences in the cell parameters on going from one morphology to the other have been explained by correlating the shape, structure and the above-described interfacial properties of a given TiO2 morphology to the said parameters. The average magnitudes of PCEs follow the order: NWs (5.96%) > NPs (4.95%) > PNPs (4.85%) > NSHs (2.5%), with the champion cell based on NWs exhibiting a PCE of 6.29%. For NWs, an optimal balance between the fast photo-excited electron injection to NWs at the NW/CdS interface, the high resistance offered at the TiO2 NW/CdS/S(2-) interfaces to electron recombination with the oxidized electrolyte or with the holes in CdS, the low electron transport resistance in NWs, and low dark currents, yields the highest efficiency due to directional unhindered transport of electrons afforded by the NWs. For NSHs, electron trapping in the two dimensional sheets, and a high electron recombination rate prevent the effective transfer of electrons to FTO, thus reducing short circuit current density significantly, resulting in a poor performance. This study provides a deep understanding of charge transfer, transport and collection processes necessary for the design of efficient QDSCs.

  5. Segment-specific Ca(2+) transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages.

    Science.gov (United States)

    Browne, Austin; O'Donnell, Michael J

    2016-04-01

    Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca(2+) transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca(2+) transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca(2+) fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca(2+) regulation. Basolateral Ca(2+) transport across distal tubules of Drosophila varied throughout the life cycle; Ca(2+) was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾ 168 h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca(2+) uptake and Ca(2+) release across the basolateral surface of the distal segment of the same tubule, indicating that Ca(2+) transport is bidirectional. Ca(2+) uptake across the distal segment of tubules of young adults and Ca(2+) release across the distal segment of tubules of older adults was also suggestive of reversible Ca(2+) storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca(2+) sequestration during periods of high dietary calcium intake and passive Ca(2+) release during periods of calcium deficiency.

  6. Aluminum-Doped Zinc Oxide as Highly Stable Electron Collection Layer for Perovskite Solar Cells.

    Science.gov (United States)

    Zhao, Xingyue; Shen, Heping; Zhang, Ye; Li, Xin; Zhao, Xiaochong; Tai, Meiqian; Li, Jingfeng; Li, Jianbao; Li, Xin; Lin, Hong

    2016-03-01

    Although low-temperature, solution-processed zinc oxide (ZnO) has been widely adopted as the electron collection layer (ECL) in perovskite solar cells (PSCs) because of its simple synthesis and excellent electrical properties such as high charge mobility, the thermal stability of the perovskite films deposited atop ZnO layer remains as a major issue. Herein, we addressed this problem by employing aluminum-doped zinc oxide (AZO) as the ECL and obtained extraordinarily thermally stable perovskite layers. The improvement of the thermal stability was ascribed to diminish of the Lewis acid-base chemical reaction between perovskite and ECL. Notably, the outstanding transmittance and conductivity also render AZO layer as an ideal candidate for transparent conductive electrodes, which enables a simplified cell structure featuring glass/AZO/perovskite/Spiro-OMeTAD/Au. Optimization of the perovskite layer leads to an excellent and repeatable photovoltaic performance, with the champion cell exhibiting an open-circuit voltage (Voc) of 0.94 V, a short-circuit current (Jsc) of 20.2 mA cm(-2), a fill factor (FF) of 0.67, and an overall power conversion efficiency (PCE) of 12.6% under standard 1 sun illumination. It was also revealed by steady-state and time-resolved photoluminescence that the AZO/perovskite interface resulted in less quenching than that between perovskite and hole transport material.

  7. The Maf factor Traffic jam both enables and inhibits collective cell migration in Drosophila oogenesis.

    Science.gov (United States)

    Gunawan, Felix; Arandjelovic, Mimi; Godt, Dorothea

    2013-07-01

    Border cell cluster (BCC) migration in the Drosophila ovary is an excellent system to study the gene regulatory network that enables collective cell migration. Here, we identify the large Maf transcription factor Traffic jam (Tj) as an important regulator of BCC migration. Tj has a multifaceted impact on the known core cascade that enables BCC motility, consisting of the Jak/Stat signaling pathway, the C/EBP factor Slow border cells (Slbo), and the downstream effector DE-cadherin (DEcad). The initiation of BCC migration coincides with a Slbo-dependent decrease in Tj expression. This reduction of Tj is required for normal BCC motility, as high Tj expression strongly impedes migration. At high concentration, Tj has a tripartite negative effect on the core pathway: a decrease in Slbo, an increase in the Jak/Stat inhibitor Socs36E, and a Slbo-independent reduction of DEcad. However, maintenance of a low expression level of Tj in the BCC during migration is equally important, as loss of tj function also results in a significant delay in migration concomitant with a reduction of Slbo and consequently of DEcad. Taken together, we conclude that the regulatory feedback loop between Tj and Slbo is necessary for achieving the correct activity levels of migration-regulating factors to ensure proper BCC motility.

  8. Optimized pH method for DNA elution from buccal cells collected in Whatman FTA cards.

    Science.gov (United States)

    Lema, Carolina; Kohl-White, Kendra; Lewis, Laurie R; Dao, Dat D

    2006-01-01

    DNA is the most accessible biologic material for obtaining information from the human genome because of its molecular stability and its presence in every nucleated cell. Currently, single nucleotide polymorphism genotyping and DNA methylation are the main DNA-based approaches to deriving genomic and epigenomic disease biomarkers. Upon the discontinuation of the Schleicher & Schuell IsoCode product (Dassel, Germany), which was a treated paper system to elute DNA from several biologic sources for polymerase chain reaction (PCR) analysis, a high-yielding DNA elution method was imperative. We describe here an improved procedure of the not fully validated Whatman pH-based elution protocol. Our DNA elution procedure from buccal cells collected in Whatman FTA cards (Whatman Inc., Florham Park, NJ) yielded approximately 4 microg of DNA from a 6-mm FTA card punch and was successfully applied for HLA-DQB1 genotyping. The genotypes showed complete concordance with data obtained from blood of the same subjects. The achieved high DNA yield from buccal cells suggests a potential cost-effective tool for genomic and epigenomic disease biomarkers development.

  9. Evaluation of ink-jet printed current collecting grids and bushbars for ITO-free organic solar cells

    NARCIS (Netherlands)

    Galagan, Y.O.; Coenen, E,W.C.; Sabik, S.; Gorter, H.H.; Barink, M.; Veenstra, S.C.; Kroon, J.M.; Andriessen, H.A.J.M.; Blom, P.W.M.

    2012-01-01

    ITO-free organic solar cells with ink-jet printed current collecting grids and high conducting PEDOT:PSS as composite anode are demonstrated. Inkjet printed current collecting grids with different cross-sectional are as have been investigated. The effect of the width and height of the gridlines and

  10. Evaluation of ink-jet printed current collecting grids and bushbars for ITO-free organic solar cells

    NARCIS (Netherlands)

    Galagan, Y.O.; Coenen, E,W.C.; Sabik, S.; Gorter, H.H.; Barink, M.; Veenstra, S.C.; Kroon, J.M.; Andriessen, H.A.J.M.; Blom, P.W.M.

    2012-01-01

    ITO-free organic solar cells with ink-jet printed current collecting grids and high conducting PEDOT:PSS as composite anode are demonstrated. Inkjet printed current collecting grids with different cross-sectional are as have been investigated. The effect of the width and height of the gridlines and

  11. Application of the collection probability of a solar cell for extracting recombination parameters from the spectral response

    Energy Technology Data Exchange (ETDEWEB)

    Tuominen, E.; Yli-Koski, M.; Haerkoenen, J.; Sinkkonen, J.

    1998-12-31

    Getting reliable information about charge carrier recombination properties is extremely important in monitoring and controlling solar cell fabrication processes. We have developed a mathematical method to extract solar cell parameters, such as diffusion lengths and surface recombination velocities directly from the spectral response of a solar cell by inverse Laplace transform. In this paper we present an improved analytical calculation of the explicit formula for the theoretical collection probability function of a solar cell. Further, we apply the obtained function in order to extract solar cell recombination parameters from spectral responses measured from solar cells processed in our laboratory. (orig.) 20 refs.

  12. Histochemical study of the oviducal gland and analysis of the sperm storage tubules of Mustelus schmitti Springer, 1939 (Chondrichthyes, Triakidae

    Directory of Open Access Journals (Sweden)

    Fernanda Gabriela Elías

    2015-08-01

    Full Text Available Abstract: The paired oviducal glands of immature and mature females of Mustelus schmitti were examined macro and microscopically. Findings indicate that these glands possessed the same zonation as in most chondrichthyans from anterior to posterior: club, papillary, baffle and terminal zones. The whole gland is composed by simple tubular glands that connect with transverse grooves all along the organ. The club zone presents a typical indian club shape with a simple columnar and ciliated epithelium including secretory cells PAS (+ and AB (+. The papillary zone is characterized by lamella forming small and long cones in numbers of three. The epithelium of this zone contains ciliated cells with apical nuclei and secretory cells with basal nuclei that stain AB (+The baffle zone consists of apically flattened lamellae alternating with spinnerets which are small projections disposed by both sides of the plateau. This whole structure is present in number of 8 or 9 units. A simple columnar ciliated epithelium covers the plateau and spinnerets and no AB or PAS staining is observed. The epithelium of the terminal zone is PAS (- and AB (+, and elongated tubules, that run adjacent to the baffle zone are the site where groups of spermatozoa are clearly observed in the lumen. The epithelium of the sperm storage tubules do not stain with any of the dyes tested. Sperm was also observed in the baffle zone, presumably in its way to the fecundation in the oviduct because it displays no aggregation pattern and was between the folds of the epithelium. By scanning electron microscopy sperm was observed in the club and baffle zones in a gland which belonged to a pregnant female.

  13. Continuous Collection of Adeno-Associated Virus from Producer Cell Medium Significantly Increases Total Viral Yield.

    Science.gov (United States)

    Benskey, Matthew J; Sandoval, Ivette M; Manfredsson, Fredric P

    2016-02-01

    The ability to efficiently produce large amounts of high-titer recombinant adeno-associated virus (AAV) is a prerequisite to the continued success of AAV as a gene therapy tool targeted toward large-animal preclinical studies or human clinical therapeutics. Current manufacturing procedures necessitate laborious and time-consuming purification procedures to obtain AAV particles of sufficient titer and purity for these demanding biomedical applications. The finding that AAV can be harvested and purified from producer cell medium may represent an efficient alternative to purifying AAV from cellular lysates. Here we sought to determine the maximum duration of time, and frequency within which AAV can be harvested from producer cell medium, in order to maximize the yield obtained from a single transfection preparation. Human embryonic kidney 293T cells were transfected with polyethylenimine to produce AAV2/5 expressing green fluorescent protein (GFP), and cellular medium was harvested every 2 days until a maximum duration of 19 days posttransfection. AAV2/5-GFP was released into producer cell medium at a steady state until 7 days posttransfection, at which time titers dropped dramatically. Harvesting medium every two days resulted in the maximum yield of AAV from a single preparation, and the cumulative yield of AAV harvested from the producer cell medium was 4-fold higher than the yield obtained from a traditional purification of AAV from cellular lysates. The AAV2/5 harvested from medium within the 7-day collection time-course mediated high levels of transduction in vivo, comparable to AAV2/5 harvested from cellular lysates. AAV purified from cell lysates showed increasing amounts of empty particles at 5 and 7 days posttransfection, whereas AAV purified from cell medium did not show an increase in the amount of empty particles throughout the 7-day time course. Finally, we extended these findings to AAV2/9, demonstrating that a comparable ratio of AAV2/9 particles are

  14. [The assessment of the regularity of the nephron anlage tubule formation on the basis of provisionality principle].

    Science.gov (United States)

    Panteleev, S M; Vikhareva, L V; Mal'tseva, N G; Ushakov, A L; Khamoshina, I Ia; Iaroslavtseva, O F; Chivshina, R V; Pal'chenkova, N O; Margarian, A V; Belkhoroeva, M M

    2011-01-01

    The study of the definitive kidneys of 94 human embryos and fetuses at 4.5 to 12 weeks of gestation, has demonstrated that the formation of the proximal nephron tubules resulted from the cellular proliferation in the area of transition of the capsule of the renal corpuscle into the tubular part of the nephron that occurs only after the completion of the segregation of the renal corpuscle and the distal tubule within the nephron anlage. The formation of the renal tubules in the nephron anlage seems to be determined phylogenetically, while the initial differentiation of the distal tubule is a provisional feature.

  15. Collecting duct renal cell carcinoma with the syndrome of inappropriate antidiuresis: An autopsy case report

    Directory of Open Access Journals (Sweden)

    Emi Yasuda

    2013-01-01

    Full Text Available A 57-year-old Japanese man visited our hospital with a moist cough. Chest radiographic imaging showed a left hilar shadow. Adenocarcinoma cells were found on cytologic screening of fresh sputum. Although multiple metastases including brain were detected, no tumor was observed in the kidneys. The patient underwent whole-brain irradiation and chemotherapy for advanced-stage lung cancer. One month before his death, carcinomatous meningitis was detected. Hyponatremia, hypo-osmolality, and hypertonic urine suggested the syndrome of inappropriate antidiuresis. Restricting water intake improved the hyponatremia; however, he developed fever and hematuria. Despite systemic administration of an antibacterial drug, he died. Primary tumor in the lung was absent, but adenocarcinoma of the right kidney was evident on autopsy. Lectin histochemical analysis of the carcinoma revealed its distal nephron origin, confirming collecting duct carcinoma. Severe carcinomatous meningitis, which is possibly caused the syndrome of inappropriate antidiuresis, was observed, with no cancer involvement of the pituitary gland and hypothalamus.

  16. Increased expression of intranuclear matrix metalloproteinase 9 in atrophic renal tubules is associated with renal fibrosis.

    Directory of Open Access Journals (Sweden)

    Jen-Pi Tsai

    Full Text Available BACKGROUND: Reduced turnover of extracellular matrix has a role in renal fibrosis. Matrix metalloproteinases (MMPs is associated with many glomerular diseases, but the histological association of MMPs and human renal fibrosis is unclear. METHODS: This is a retrospective study. Institutional Review Board approval was obtained for the review of patients' medical records, data analysis and pathological specimens staining with waiver of informed consents. Specimens of forty-six patients were examined by immunohistochemical stain of MMP-9 in nephrectomized kidneys, and the association of renal expression of MMP-9 and renal fibrosis was determined. MMP-9 expression in individual renal components and fibrosis was graded as high or low based on MMP-9 staining and fibrotic scores. RESULTS: Patients with high interstitial fibrosis scores (IFS and glomerular fibrosis scores (GFS had significantly higher serum creatinine, lower estimated glomerular filtration rate (eGFR, and were more likely to have chronic kidney disease (CKD and urothelial cell carcinoma. Univariate analysis showed that IFS and GFS were negatively associated with normal and atrophic tubular cytoplasmic MMP-9 expression and IFS was positively correlated with atrophic tubular nuclear MMP-9 expression. Multivariate stepwise regression indicated that MMP-9 expression in atrophic tubular nuclei (r = 0.4, p = 0.002 was an independent predictor of IFS, and that MMP-9 expression in normal tubular cytoplasm (r = -0.465, p<0.001 was an independent predictor of GFS. CONCLUSIONS: Interstitial fibrosis correlated with MMP-9 expression in the atrophic tubular nuclei. Our results indicate that renal fibrosis is associated with a decline of MMP-9 expression in the cytoplasm of normal tubular cells and increased expression of MMP-9 in the nuclei of tubular atrophic renal tubules.

  17. Light Collection and Pulse-Shape Discrimination in Elongated Scintillator Cells for the PROSPECT Reactor Antineutrino Experiment

    CERN Document Server

    Ashenfelter, J; Band, H R; Barclay, G; Bass, C D; Berish, D; Bowden, N S; Bowes, A; Brodsky, J P; Bryan, C D; Cherwinka, J J; Chu, R; Classen, T; Commeford, K; Davee, D; Dean, D; Deichert, G; Diwan, M V; Dolinski, M J; Dolph, J; Dwyer, D A; Gaison, J K; Galindo-Uribarri, A; Gilje, K; Glenn, A; Goddard, B W; Green, M; Han, K; Hans, S; Heeger, K M; Heffron, B; Jaffe, D E; Langford, T J; Littlejohn, B R; Caicedo, D A Martinez; McKeown, R D; Mendenhall, M P; Mueller, P; Mumm, H P; Napolitano, J; Neilson, R; Norcini, D; Pushin, D; Qian, X; Romero, E; Rosero, R; Saldana, L; Seilhan, B S; Sharma, R; Sheets, S; Stemen, N T; Surukuchi, P T; Varner, R L; Viren, B; Wang, W; White, B; White, C; Wilhelmi, J; Williams, C; Wise, T; Yao, H; Yeh, M; Yen, Y -R; Zangakis, G; Zhang, C; Zhang, X

    2015-01-01

    A meter-long, 23-liter EJ-309 liquid scintillator detector has been constructed to study the light collection and pulse-shape discrimination performance of elongated scintillator cells for the PROSPECT reactor antineutrino experiment. The magnitude and uniformity of light collection and neutron/gamma discrimination power in the energy range of antineutrino inverse beta decay products have been studied using gamma and spontaneous fission calibration sources deployed along the cell long axis. We also study neutron-gamma discrimination and light collection abilities for differing PMT and reflector configurations. Key design features for optimizing MeV-scale response and background rejection capabilities are identified.

  18. T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases

    Directory of Open Access Journals (Sweden)

    Al-Qusairi Lama

    2011-07-01

    Full Text Available Abstract In skeletal muscle, the excitation-contraction (EC coupling machinery mediates the translation of the action potential transmitted by the nerve into intracellular calcium release and muscle contraction. EC coupling requires a highly specialized membranous structure, the triad, composed of a central T-tubule surrounded by two terminal cisternae from the sarcoplasmic reticulum. While several proteins located on these structures have been identified, mechanisms governing T-tubule biogenesis and triad formation remain largely unknown. Here, we provide a description of triad structure and plasticity and review the role of proteins that have been linked to T-tubule biogenesis and triad formation and/or maintenance specifically in skeletal muscle: caveolin 3, amphiphysin 2, dysferlin, mitsugumins, junctophilins, myotubularin, ryanodine receptor, and dihydhropyridine Receptor. The importance of these proteins in triad biogenesis and subsequently in muscle contraction is sustained by studies on animal models and by the direct implication of most of these proteins in human myopathies.

  19. Hypoxia Induces a HIF-1-Dependent Transition from Collective-to-Amoeboid Dissemination in Epithelial Cancer Cells

    NARCIS (Netherlands)

    Lehmann, S.A.; Boekhorst, V. Te; Odenthal, J.; Bianchi, R.; Helvert, S. van; Ikenberg, K.; Ilina, O.; Stoma, S.; Xandry, J.; Jiang, L.; Grenman, R.; Rudin, M.; Friedl, P.

    2017-01-01

    Cancer metastases arise from a multi-step process that requires metastasizing tumor cells to adapt to signaling input from varying tissue environments [1]. As an early metastatic event, cancer cell dissemination occurs through different migration programs, including multicellular, collective, and

  20. Effect of theobromine-containing toothpaste on dentin tubule occlusion in situ.

    Science.gov (United States)

    Amaechi, Bennett T; Mathews, Sapna M; Mensinkai, Poornima K

    2015-01-01

    Dentin hypersensitivity (DH) is treated by either occlusion of dentin tubules or nerve desensitization. This in situ study compared dentin tubules occlusion by theobromine-containing dentifrices with (Theodent-classic-F®, TCF) and without (Theodent-classic®, TC) fluoride with 1,500 ppm fluoride toothpaste, Colgate®-Regular (Fluoride) and Novamin®-containing toothpaste, Sensodyne®-5000-Nupro (Novamin®). Each subject wore four intraoral appliances bearing dentin blocks while using one of four test dentifrices (n = 20/dentifrice) twice daily for 7 days. The four appliances were removed successively after 1, 2, 3, and 7 days. Treated blocks and their control (untreated) blocks were examined with scanning electron microscopy (SEM). Effects were compared statistically (ANOVA/Tukey's) based on percentage of surface area covered by deposited precipitate layer (%DPL) and percentage of fully open (%FOT), partially occluded (%POT), and completely occluded (%COT) tubules in each block calculated relative to the number of tubules in their control blocks. SEM observation indicated an increased %COT and %DPL over time. After 1 and 2 days, %COT was comparable with TC and TCF, and significantly (p < 0.05) higher compared with Novamin® and Fluoride. Following 3 and 7 days, %COT was comparable among TC, TCF, and Novamin®, but remained significantly lower in Fluoride. At any time, %DPL was significantly (p < 0.05) higher in TC, TCF, and Novamin® compared with Fluoride. Theobromine-containing toothpastes with and without fluoride have equal potential in occluding dentin tubules within a shorter time period than Novamin®-containing toothpaste; however, the three demonstrated equal potential after 1 week, but not the fluoride toothpaste. Theobromine-containing toothpaste promoted dentin tubule occlusion thus shows potential to relief DH.

  1. Cellular localization of uranium in the renal proximal tubules during acute renal uranium toxicity.

    Science.gov (United States)

    Homma-Takeda, Shino; Kitahara, Keisuke; Suzuki, Kyoko; Blyth, Benjamin J; Suya, Noriyoshi; Konishi, Teruaki; Terada, Yasuko; Shimada, Yoshiya

    2015-12-01

    Renal toxicity is a hallmark of uranium exposure, with uranium accumulating specifically in the S3 segment of the proximal tubules causing tubular damage. As the distribution, concentration and dynamics of accumulated uranium at the cellular level is not well understood, here, we report on high-resolution quantitative in situ measurements by high-energy synchrotron radiation X-ray fluorescence analysis in renal sections from a rat model of uranium-induced acute renal toxicity. One day after subcutaneous administration of uranium acetate to male Wistar rats at a dose of 0.5 mg uranium kg(-1) body weight, uranium concentration in the S3 segment of the proximal tubules was 64.9 ± 18.2 µg g(-1) , sevenfold higher than the mean renal uranium concentration (9.7 ± 2.4 µg g(-1) ). Uranium distributed into the epithelium of the S3 segment of the proximal tubules and highly concentrated uranium (50-fold above mean renal concentration) in micro-regions was found near the nuclei. These uranium levels were maintained up to 8 days post-administration, despite more rapid reductions in mean renal concentration. Two weeks after uranium administration, damaged areas were filled with regenerating tubules and morphological signs of tissue recovery, but areas of high uranium concentration (100-fold above mean renal concentration) were still found in the epithelium of regenerating tubules. These data indicate that site-specific accumulation of uranium in micro-regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney.

  2. Ammonium hexafluorosilicate elicits calcium phosphate precipitation and shows continuous dentin tubule occlusion.

    Science.gov (United States)

    Suge, Toshiyuki; Kawasaki, Akiko; Ishikawa, Kunio; Matsuo, Takashi; Ebisu, Shigeyuki

    2008-02-01

    Diamine silver fluoride [AgF: (NH(3))(2)AgF] has been used clinically in Japan, as it reduces dental caries and dentin hypersensitivity. However, AgF stains the teeth black due to silver precipitation. To overcome this drawback, the authors prepared ammonium hexafluorosilicate [SiF: (NH(4))(2)SiF(6)], which does not stain the teeth, and SiF occluded open dentin tubules completely with silica-calcium phosphate precipitate. The aim of this study was to evaluate the duration of dentin tubule occlusion after SiF treatment in a simulated oral environment. To simulate dentin tubules subject to dentin hypersensitivity, dentin disks were treated with EDTA for 2 min. The disks were treated with 0.476 mol/L SiF for 3 min, and then the disks were immersed in synthetic saliva, which was regularly replenished to maintain its ionic concentration, for up to 7 days. The occluding ability of the dentin tubules was evaluated using scanning electron microscopy (SEM), and the hydraulic conductance was measured following Pashley's method at regular intervals. SEM photographs demonstrated that dentin tubules were occluded homogeneously and completely with the precipitate at 7 days after treatment with SiF. In addition, newly formed calcium phosphate precipitate was present at the dentin surface. The dentin permeability showed a consistently low value throughout the experimental period. The values immediately after SiF treatment and 7 days after immersion were 11.9+/-3.7% and 7.9+/-2.9%, respectively. Ammonium hexafluorosilicate is useful for the treatment of dentin hypersensitivity, since ammonium hexafluorosilicate induced calcium phosphate precipitation from the saliva; therefore, it has a continuous effect on dentin tubules occlusion under a simulated oral environment.

  3. Why and how does collective red blood cells motion occur in the blood microcirculation?

    Science.gov (United States)

    Ghigliotti, Giovanni; Selmi, Hassib; Asmi, Lassaad El; Misbah, Chaouqi

    2012-10-01

    The behaviour of red blood cells (RBCs), modelled as vesicles, in Poiseuille flow, mimicking the microvasculature, is studied with numerical simulations in two dimensions. RBCs moving in the centre of the Poiseuille flow (as in blood capillaries) are shown to attract each other and form clusters only due to hydrodynamic interactions, provided that their distance at a given time is below a certain critical value. This distance depends on physical parameters, such as the flow strength. Our simulations reveal that clusters are unstable above a threshold value in the number of forming RBCs, beyond which one or few cells escape the pack by a self-regulating mechanism that select the marginally stable size. This size selection depends on the flow strength as well as on the RBC swelling ratio. The results are interpreted via the analysis of the perturbation of the flow field induced by the vesicles and the interplay with bending and tension forces. This sheds a novel light on the process of collective motion of RBCs observed in vivo.

  4. 5-Lypoxygenase products are involved in renal tubulointerstitial injury induced by albumin overload in proximal tubules in mice.

    Directory of Open Access Journals (Sweden)

    Sharon Schilling Landgraf

    Full Text Available The role of albumin overload in proximal tubules (PT in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type and 5-lipoxygenase-deficient mice (5-LO(-/-. The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO(-/- mice. The levels of urinary protein observed in the 5-LO(-/- mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO(-/- mice. LTB4 and LTD4, 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-α and interleukin (IL-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO(-/- mice. However, 5-LO(-/- mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload.

  5. 5-Lypoxygenase products are involved in renal tubulointerstitial injury induced by albumin overload in proximal tubules in mice.

    Science.gov (United States)

    Landgraf, Sharon Schilling; Silva, Leandro Souza; Peruchetti, Diogo Barros; Sirtoli, Gabriela Modenesi; Moraes-Santos, Felipe; Portella, Viviane Gomes; Silva-Filho, João Luiz; Pinheiro, Carla Silva; Abreu, Thiago Pereira; Takiya, Christina Maeda; Benjamin, Claudia Farias; Pinheiro, Ana Acacia Sá; Canetti, Claudio; Caruso-Neves, Celso

    2014-01-01

    The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO(-/-)). The changes in glomerular morphology and nestin expression observed in w