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Sample records for bovine chromaffin cells

  1. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

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    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  2. Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

    DEFF Research Database (Denmark)

    Cheek, T R; Thastrup, Ole

    1989-01-01

    Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3......+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist...

  3. Characterization of a novel, hydrophilic dihydropyridine, NKY-722, as a Ca2+ antagonist in bovine cultured adrenal chromaffin cells.

    OpenAIRE

    Ohue, T.; Lee, K; Koshimura, K.; Miwa, S

    1991-01-01

    1. To characterize NKY-722, a novel hydrophilic dihydropyridine derivative, as a Ca2+ antagonist, we examined its effects on 45Ca2+ influx, intracellular free Ca2+ concentrations [( Ca2+]i), and release of noradrenaline and adrenaline in bovine cultured adrenal chromaffin cells. 2. NKY-722 had little effect on basal 45Ca2+ influx into the resting cells, but inhibited high K+ (35.9 mM)-evoked 45Ca2+ influx in a concentration-dependent manner with an IC50 value of 5.2 nM. 3. NKY-722 inhibited h...

  4. Xenotransplantation of microencaps bovine chromaffin cells into hemiparkinsonian monkeys:a analyses of behaviour,biochemistry and pathology

    Institute of Scientific and Technical Information of China (English)

    XUE Y. L.; WANG L. N.; WANG Z. F.; ZHONG D. G.; LI X. J.; CUI X.; MA X. J.; ZHU Ming-wei; SUN A. M.

    2001-01-01

    @@ This study examines the effects of xenografts of microencapsulated bovine chromaffin cells (BCCs) on the rotational behavior of hemiparkinsonian monkey recipients. In addition, it determines the content of monoamine neurotransmitters and their major metabolites in the neostriatum in hemiparkinsonian monkeys. The hemiparkinsonian model in monkeys was induced by a unilateral intracarotid injection of methyl-phenyl-tetrahydropyridine (MPTP). Unencapsulated BCCs (n = 2), BCCs microencapsulated (n= 6) in alginate-polylysine-alginate (APA) membranes as well as empty microencapsules (n = 1) were grafted into the neostriatum of the hemiparkinsonian monkeys.

  5. Effects of inorganic mercury (Hg{sup 2+}) on calcium channel currents and catecholamine release from bovine chromaffin cells

    Energy Technology Data Exchange (ETDEWEB)

    Weinsberg, F. [Medical Inst. of Environmental Hygiene, Heinrich Heine University, Duesseldorf (Germany); Bickmeyer, U. [Medical Inst. of Environmental Hygiene, Heinrich Heine University, Duesseldorf (Germany); Wiegand, H. [Medical Inst. of Environmental Hygiene, Heinrich Heine University, Duesseldorf (Germany)

    1995-01-01

    The effects of Hg{sup 2+} on calcium channel currents and the potassium-evoked catecholamine release of bovine chromaffin cells in culture were examined. The effects of Cd{sup 2+} were studied for comparison. Calcium channel currents were recorded in the whole-cell configuration of the patch-clamp technique. In a concentration of 100 {mu}M, Hg{sup 2+} blocked the currents completely; 100 {mu}M Cd{sup 2+} had the same effect. Potassium-evoked catecholamine release from chromaffin cells was measured at different timepoints with HPLC under control conditions and in the presence of different Hg{sup 2+} concentrations. Low Hg{sup 2+} concentrations (0.1 and 1 {mu}M) did not affect the amount of the catecholamines epinephrine (E) and norepinephrine (NE) which was released. Under identical conditions 1 {mu}M Cd{sup 2+} also had no effect on release. With 10 {mu}M Hg{sup 2+} there was a time-dependent increase in the potassium-evoked catecholamine release (by 27% after 8 min). The E/NE ratio was not altered. In contrast to this, the release was slightly reduced with 10 {mu}M Cd{sup 2+}. In the presence of 100 {mu}M Hg{sup 2+}, there was a reduction of the release during an early phase, followed by an increase. The calcium channel block by 100 {mu}M Cd{sup 2+} also reduced the release significantly. Catecholamine release of bovine chromaffin cells is driven into two opposite directions by Hg{sup 2+}. On the one hand, a calcium channel block reduces the release, while on the other hand effects occur which can increase the release. Both tendencies occur simultaneously, but have different concentration- and time-dependencies. The catecholamine output at a given timepoint reflects the `sum` of these different effects. (orig.)

  6. Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

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    Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

    1998-03-01

    Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

  7. Brevenal inhibits pacific ciguatoxin-1B-induced neurosecretion from bovine chromaffin cells.

    Directory of Open Access Journals (Sweden)

    César Mattei

    Full Text Available Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and beta-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.

  8. Trifluoperazine reduces inward ionic currents and secretion by separate mechanisms in bovine chromaffin cells.

    Science.gov (United States)

    Clapham, D E; Neher, E

    1984-08-01

    Using patch-clamp techniques, excitation and secretion in chromaffin cells were studied by measurement of unitary inward currents and of stimulus-evoked increments in membrane capacitance. The effect of the calmodulin inhibitor trifluoperazine (TFP) on Na, Ca and acetylcholine-induced (ACh) currents as well as on capacitance increments was investigated. TFP in concentrations up to 10 microM had no effect on Na channel currents. TFP was a potent anticholinergic agent. TFP in concentrations of 100 nM-1 microM decreased net ACh-induced currents by a slow block or allosteric modification of the channel. The effect was only partially reversible. Recovery from desensitization was retarded in direct relation to [TFP]. At the single channel level, TFP was found to slightly shorten open times in 0.5 and 20 microM-ACh. As reported previously, desensitization can be modelled by at least two desensitized states, as reflected by the bursting and clustering behaviour of single channels. TFP shortened clusters mainly by reducing the number of bursts per cluster. Whole-cell Ca currents (ICa) were reduced in 10 microM-TFP from an average of 29 microA cm-2-13 microA cm-2. Changes in capacitance of 1-200 fF were elicited in controls by maximal activation of the Ca current. We interpreted these steps to be the summed result of many exocytotic vesicular fusion events. Capacitance steps depended on ICa and were absent when extracellular Ca was removed. Application of 10 microM-TFP inhibited capacitance steps. The block of capacitance steps by TFP was shown to be independent of the reduction of ACh and Ca inward ionic currents. We conclude that the prevention of exocytosis by TFP is not completely described by its inhibition of electrical excitability but also results from intracellular actions. PMID:6090644

  9. Effects of collagenase on the release of [3H]-noradrenaline from bovine cultured adrenal chromaffin cells.

    Science.gov (United States)

    Almazan, G.; Aunis, D.; García, A. G.; Montiel, C.; Nicolás, G. P.; Sánchez-García, P.

    1984-01-01

    Bovine isolated adrenal chromaffin cells maintained in culture at 37 degrees C for 1-7 days become polygonal and bipolar, with typical varicosity-like extensions. Catecholamine levels and dopamine beta-hydroxylase activity decreased after 24-48 h of culture, but recovered to normal levels 3-7 days later. Incubation of 1-7 day-old cells in the presence of increasing concentrations of [3H]-noradrenaline (3.91 to 125 nM) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day-old cells took up and retained only one third of the radioactivity found in 2-7 day-old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mM K+ was very poor in 24 h-old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2-7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatment. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor-mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage-sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by

  10. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.

    OpenAIRE

    Neher, E; Marty, A.

    1982-01-01

    The capacitance of the surface membrane of small adrenal chromaffin cells was measured with patch-clamp pipettes. Continuous and discrete changes of capacitance were observed. They were interpreted as changes of surface area connected to exocytotic or endocytotic processes. Most of the measurements were performed in the "whole-cell" recording configuration [Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100], which allows the intracellular Ca2...

  11. Dissociation of Ca sup 2+ entry and Ca sup 2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

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    Stauderman, K.A.; Pruss, R.M. (Merrell Dow Research Institute, Cincinnati, OH (USA))

    1989-11-05

    In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium (( Ca2+)i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of (3H)inositol 1,4,5-trisphosphate (( 3H)Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ (( Ca2+)o) was less than 100 nM, AII still rapidly increased (Ca2+)i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When (Ca2+)o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused (Ca2+)i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low (Ca2+)o, ionomycin pretreatment abolished both the (Ca2+)i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of (Ca2+)i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of (3H)Ins(1,4,5)P3. We conclude that AII increased (Ca2+)i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.

  12. Uptake of meta-iodobenzylguanidine by bovine chromaffin granule membranes

    International Nuclear Information System (INIS)

    meta-Iodobenzylguanidine, an adrenal imaging agent used for the scintigraphic detection of human pheochromocytoma, is a substrate for the monoamine uptake system of chromaffin granules. It is accumulated by bovine chromaffin granule membrane vesicles in the presence of ATP, and it can be released by an osmotic shock. The uptake is dependent upon the generation of an H+-electrochemical gradient by an ATP-dependent H+ pump since it is blocked by an H+ ionophore and since meta-iodobenzylguanidine uptake can be driven by imposing an artificial pH gradient (inside acidic) on the membrane vesicles. The transport is saturable and its Km value (2.0 microM at pH 8.0) is similar to that of noradrenaline (5.3 microM). Transport occurs through the monoamine transporter since it is blocked by the same inhibitors, tetrabenazine and reserpine, and also by the transporter substrates noradrenaline and serotonin. Noradrenaline inhibits meta-iodobenzylguanidine uptake competitively (Ki = 13 microM). In addition, meta-iodobenzylguanidine displaces dihydrotetrabenazine and reserpine from their binding sites on chromaffin granule membranes. It is thus likely that, after in vivo administration, [131I] meta-iodobenzylguanidine is ultimately stored in chromaffin granules and that it is translocated by the monoamine transporter

  13. Dopamine receptors on adrenal chromaffin cells modulate calcium uptake and catecholamine release

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    Bigornia, L.; Suozzo, M.; Ryan, K.A.; Napp, D.; Schneider, A.S.

    1988-10-01

    The presence of dopamine-containing cells in sympathetic ganglia, i.e., small, intensely fluorescent cells, has been known for some time. However, the role of dopamine as a peripheral neurotransmitter and its mechanism of action are not well understood. Previous studies have demonstrated the presence of D2 dopamine receptors on the surface of bovine adrenal chromaffin cells using radioligand binding methods and dopamine receptor inhibition of catecholamine release from perfused adrenal glands. In the present study, we provide evidence confirming a role of dopamine receptors as inhibitory modulators of adrenal catecholamine release from bovine chromaffin cell cultures and further show that the mechanism of modulation involves inhibition of stimulated calcium uptake. Apomorphine gave a dose-dependent inhibition (IC50 = 1 microM) of 45Ca2+ uptake stimulated by either nicotine (10 microM) or membrane depolarization with an elevated K+ level (60 mM). This inhibition was reversed by a series of specific (including stereospecific) dopamine receptor antagonists: haloperidol, spiperone, sulpiride, and (+)-butaclamol, but not (-)-butaclamol. In addition, the calcium channel agonist Bay K 8644 was used to stimulate uptake of 45Ca2+ into chromaffin cells, and this uptake was also inhibited by the dopamine receptor agonist apomorphine. The combined results suggest that dopamine receptors on adrenal chromaffin cells alter Ca2+ channel conductance, which, in turn, modulates catecholamine release.

  14. Subcellular compartmentalization of 1-methyl-4-phenylpyridinium with catecholamines in adrenal medullary chromaffin vesicles may explain the lack of toxicity to adrenal chromaffin cells

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    Reinhard, J.F. Jr.; Diliberto, E.J. Jr.; Viveros, O.H.; Daniels, A.J.

    1987-11-01

    Cultures of bovine adrenomedullary chromaffin cells accumulated 1-methyl-4-phenylpyridinium (MPP/sup +/) in a time- and concentration-dependent manner by a process that was prevented by desmethylimipramine. The subcellular localization of the incorporated (methyl-/sup 3/H)MPP/sup +/ was examined by differential centrifugation and sucrose density gradient fractionation and was found to be predominantly colocalized with catecholamines in chromaffin vesicles, and negligible amounts were detected within the mitochondrial fraction. When chromaffin cell membranes were made permeable with the detergent digitonin the absence of calcium, there was no increase in the release of (/sup 3/H)MPP/sup +/, indicating that there is negligible accumulation of the neurotoxin in the cytosol. Simultaneous exposure to digitonin and calcium induced cosecretion of MPP/sup +/ and catecholamines. Stimulation of the cells with nicotine released both catecholamines and MPP/sup +/ at identical rates and percentages of cellular content in a calcium-dependent manner. Last, when cells were incubated with MPP/sup +/ in the presence of tetrabenazine (an inhibitor of vesicular uptake), the chromaffin cell toxicity of MPP/sup +/ was potentiated. The authors submit that the ability of the chromaffin cells to take up and store MPP/sup +/ in the chromaffin vesicle prevents the toxin's interaction with other structures and, thus, prevents cell damage. As an extension of this hypothesis, the relative resistance of some brain monoaminergic neurons to the toxic actions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine may result from the subcellular sequestration of MPP/sup +/ in the storage vesicle.

  15. Computing the chromaffin cell: a research-community curator/user approach to biocomputation for chromaffin cell biology.

    Science.gov (United States)

    Eiden, Lee E; Hirsch, Michael D

    2002-10-01

    Exocytosis, stimulus-secretion coupling, real-time measurements of neurosecretion, and stimulus-secretion-synthesis coupling (stimulus-transcription coupling) were all initially proposed and verified in the chromaffin cell. Detailed analysis of the molecules and pathways responsible for secretion and transsynaptic regulation of gene expression patterns in neuroendoccrine cells have been very fruitfully explored in chromaffin cells and the related PC12 pheochromocytoma cell line, using modern molecular biologcal, cellular imaging, and expression profiling techniques. The time is clearly at hand for a concerted bioinformatics approach to acquiring and synthesizing electrophysiological, biochemical, and proteomic/genomic data on the chromaffin cell. Accelerating this process will fully realize the unique attributes of the chromaffin cell as a homogeneous, accessible, fully functional model of the posttmitotic neuroendocrine cell.

  16. Vesicle Pools: Lessons from Adrenal Chromaffin Cells

    Directory of Open Access Journals (Sweden)

    David R Stevens

    2011-02-01

    Full Text Available The adrenal chromaffin cell serves as a model system to study fast Ca2+-dependent exocytosis. Membrane capacitance measurements in combination with Ca2+ uncaging offers a temporal resolution in the millisecond range and reveals that catecholamine release occurs in three distinct phases. Release of a readily releasable (RRP and a slowly releasable (SRP pool are followed by sustained release, due to maturation and release of vesicles which were not release-ready at the start of the stimulus. Trains of depolarizations, a more physiological stimulus, induce release from a small immediately releasable pool of vesicles residing adjacent to calcium channels, as well as from the RRP. The SRP is poorly activated by depolarization. A sequential model, in which non-releasable docked vesicles are primed to a slowly releasable state, and then further mature to the readily releasable state, has been proposed. The docked state, dependent on membrane proximity, requires SNAP-25, synaptotagmin and syntaxin. The ablation or modification of SNAP-25 and syntaxin, components of the SNARE complex, as well as of synaptotagmin, the calcium sensor, and modulators such complexins and Snapin alter the properties and/or magnitudes of different phases of release, and in particular can ablate the RRP. These results indicate that the composition of the SNARE complex and its interaction with modulatory molecules drives priming and provides a molecular basis for different pools of releasable vesicles.

  17. Platelet granule exocytosis: A comparison with chromaffin cells

    Directory of Open Access Journals (Sweden)

    Jennifer eFitch-Tewfik

    2013-06-01

    Full Text Available The rapid secretion of bioactive amines from chromaffin cells constitutes an important component of the fight or flight response of mammals to stress. Platelets respond to stresses within the vasculature by rapidly secreting cargo at sites of injury, inflammation, or infection. Although chromaffin cells derive from the neural crest and platelets from bone marrow megakaryocytes, both have evolved a heterogeneous assemblage of granule types and a mechanism for efficient release. This article will provide an overview of granule formation and exocytosis in platelets with an emphasis on areas in which the study of chromaffin cells has influenced that of platelets and on similarities between the two secretory systems. Commonalities include the use of transporters to concentrate bioactive amines and other cargos into granules, the role of cytoskeletal remodeling in granule exocytosis, and the use of granules to provide membrane for cytoplasmic projections. The SNAREs and SNARE accessory proteins used by each cell type will also be considered. Finally, we will discuss the newly appreciated role of dynamin family proteins in regulated fusion pore formation. This evaluation of the comparative cell biology of regulated exocytosis in platelets and chromaffin cells demonstrates a convergence of mechanisms between two disparate cell types both tasked with responding rapidly to physiological stimuli.

  18. GPCRs of adrenal chromaffin cells & catecholamines: The plot thickens.

    Science.gov (United States)

    Lymperopoulos, Anastasios; Brill, Ava; McCrink, Katie A

    2016-08-01

    The circulating catecholamines (CAs) epinephrine (Epi) and norepinephrine (NE) derive from two major sources in the whole organism: the sympathetic nerve endings, which release NE on effector organs, and the chromaffin cells of the adrenal medulla, which are cells that synthesize, store and release Epi (mainly) and NE. All of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla. The secretion of CAs from adrenal chromaffin cells is regulated in a complex way by a variety of membrane receptors, the vast majority of which are G protein-coupled receptors (GPCRs), including adrenergic receptors (ARs), which act as "presynaptic autoreceptors" in this regard. There is a plethora of CA-secretagogue signals acting on these receptors but some of them, most notably the α2ARs, inhibit CA secretion. Over the past few years, however, a few new proteins present in chromaffin cells have been uncovered to participate in CA secretion regulation. Most prominent among these are GRK2 and β-arrestin1, which are known to interact with GPCRs regulating receptor signaling and function. The present review will discuss the molecular and signaling mechanisms by which adrenal chromaffin cell-residing GPCRs and their regulatory proteins modulate CA synthesis and secretion. Particular emphasis will be given to the newly discovered roles of GRK2 and β-arrestins in these processes and particular points of focus for future research will be highlighted, as well.

  19. GPCRs of adrenal chromaffin cells & catecholamines: The plot thickens.

    Science.gov (United States)

    Lymperopoulos, Anastasios; Brill, Ava; McCrink, Katie A

    2016-08-01

    The circulating catecholamines (CAs) epinephrine (Epi) and norepinephrine (NE) derive from two major sources in the whole organism: the sympathetic nerve endings, which release NE on effector organs, and the chromaffin cells of the adrenal medulla, which are cells that synthesize, store and release Epi (mainly) and NE. All of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla. The secretion of CAs from adrenal chromaffin cells is regulated in a complex way by a variety of membrane receptors, the vast majority of which are G protein-coupled receptors (GPCRs), including adrenergic receptors (ARs), which act as "presynaptic autoreceptors" in this regard. There is a plethora of CA-secretagogue signals acting on these receptors but some of them, most notably the α2ARs, inhibit CA secretion. Over the past few years, however, a few new proteins present in chromaffin cells have been uncovered to participate in CA secretion regulation. Most prominent among these are GRK2 and β-arrestin1, which are known to interact with GPCRs regulating receptor signaling and function. The present review will discuss the molecular and signaling mechanisms by which adrenal chromaffin cell-residing GPCRs and their regulatory proteins modulate CA synthesis and secretion. Particular emphasis will be given to the newly discovered roles of GRK2 and β-arrestins in these processes and particular points of focus for future research will be highlighted, as well. PMID:26851510

  20. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium

    OpenAIRE

    1982-01-01

    The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secr...

  1. Identification of a Munc13-sensitive step in chromaffin cell large dense-core vesicle exocytosis

    DEFF Research Database (Denmark)

    Man, Kwun-Nok Mimi; Imig, Cordelia; Walter, Alexander M;

    2015-01-01

    using chromaffin cells lacking individual isoforms. We show that particularly Munc13-2 plays a fundamental role in LDCV exocytosis, but in contrast to synapses lacking Munc13s, the corresponding chromaffin cells do not exhibit a vesicle docking defect. We further demonstrate that ubMunc13-2 and Munc13...

  2. Uptake of [3H]-nicotine and [3H]-noradrenaline by cultured chromaffin cells.

    Science.gov (United States)

    Ceña, V.; García, A. G.; Montiel, C.; Sánchez-García, P.

    1984-01-01

    Three day-old cultured bovine adrenal chromaffin cells incubated at room temperature with Krebs-HEPES solution containing different concentrations of [3H]-nicotine, took up and retained increasing amounts of the drug by a mechanism that did not saturate. Concentrations of cold nicotine as high as 100 microM did not alter the amount of [3H]-nicotine retained by cells. Imipramine, cocaine, tetracaine or mecamylamine, at concentrations (10 microM) that blocked the catecholamine secretory effects of nicotine completely, did not modify the uptake of [3H]-nicotine. Both imipramine and cocaine drastically inhibited [3H]-noradrenaline uptake by cells in a concentration-dependent manner (IC50S of 0.08 and 1 microM, respectively). These data indicate that the secretory effects of nicotine are not coupled to its previous uptake into cells, and are evidence in favour of a site of action for nicotine located in or at the surface of the chromaffin cell membrane. PMID:6704577

  3. The chromaffin cells of Siren lacertina (Amphibia, Urodela): cytological characteristics and evidence of exocytosis.

    OpenAIRE

    Accordi, F

    1988-01-01

    In the adrenal gland of Siren lacertina three types of chromaffin cells are described, on the basis of size, shape and electron density of the cytoplasmic granules: adrenaline-secreting cells, noradrenaline-secreting cells and small granulated chromaffin-cells. In A-cells exocytotic profiles are described, in which the granule membrane fuses with the plasma membrane and the granule core is discharged into the intercellular space.

  4. Congener specific effects by polychlorinated biphenyls on catecholamine content and release in chromaffin cells

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    Messeri, M.D. [Medizinisches Institut fuer Umwelthygiene an der Heinrich-Heine-Univ., Duesseldorf (Germany); Bickmeyer, U. [Medizinisches Institut fuer Umwelthygiene an der Heinrich-Heine-Univ., Duesseldorf (Germany); Weinsberg, F. [Physiologisches Institut, Universitaetskrankenhaus Hamburg Eppendorf, Martinistasse 52, D-20246 Hamburg (Germany); Wiegand, H. [Medizinisches Institut fuer Umwelthygiene an der Heinrich-Heine-Univ., Duesseldorf (Germany)

    1997-05-01

    The effects of the non-planar polychlorinated biphenyl (PCB) congener 2,2`,4,4`-tetrachlorobiphenyl (2,4-TCB) and of the coplanar PCB congener 3,3`,4,4`-tetrachlorobiphenyl (3,4-TCB) were investigated on the catecholamine content and release from bovine adrenal chromaffin cells in culture. Each congener was tested at three concentrations (20, 50 and 100 {mu}M) and two exposure periods (24 h and 5 days). Catecholamine release induced by K{sup +}-stimulation as well as catecholamine content of Triton X-100 treated cell cultures were examined using high-performance liquid chromatography (HPLC). 2,4-TCB showed dose- and time-dependent effects. 2,4-TCB at 100 {mu}M reduced the K{sup +}-stimulated catecholamine release after 24 h of exposure. After 5 days of exposure, 2,4 TCB at 50 and 100 {mu}M drastically reduced the K{sup +}-stimulated catecholamine release. 3,4-TCB even at a concentration of 100 {mu}M over exposure of either 24 h or 5 days had no effects on the K{sup +}-stimulated secretion. When chromaffin cells, exposed to 2,4-TCB, were lysed with 0.5% Triton X-100, a dose- and time-dependent reduction of the catecholamine content appeared. The 3,4-TCB did not reduce the catecholamine content. Conversely there seemed to be a trend towards an increase in catecholamine content. Spontaneous release of catecholamines was strongly increased by the non-planar 2,4 TCB, while the coplanar 3,4 TCB showed no effects on this parameter. Furthermore, the effects of 2,4 TCB appeared to be reversible after replacing the highest concentration (100 {mu}M) of the TCB-solution with culture-medium at the end of the 24-h exposure. Thus, K{sup +}-stimulated catecholamine release and the catecholamine content of bovine adrenal chromaffin cells was effectively reduced by the non-planar PCB congener whereas spontaneous catecholamine release was strongly increased. The coplanar PCB congener was ineffective at the same conditions. (orig.). With 5 figs., 2 tabs.

  5. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    Science.gov (United States)

    Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

    2015-01-01

    Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

  6. Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

    Directory of Open Access Journals (Sweden)

    Daungruthai Jarukanont

    Full Text Available Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We

  7. Nonreutilizaton of adrenal chromaffin granule membranes following secretion

    International Nuclear Information System (INIS)

    The intracellular postexocytotic fate of the adrenal chromaffin granule membrane (reutilization vs. nonreutilization) was addressed through two experimental approaches. First, (3H) leucine pulse-chase labeling experiments were conducted in two systems - the isolated retrograde perfused cat adrenal gland and cultured bovine adrenal chromaffin cells to compare chromaffin granule soluble dopamine-B-hydroxylase (DBH) turnover (marker for granule soluble content turnover) to that of membrane-bound DBH (marker for granule membrane turnover). Experiments in cat adrenal glands showed that at all chase periods the granule distribution of radiolabeled DBH was in agreement with the DBH activity distribution (73% membrane-bound/27% soluble) - a result consistent with parallel turnover of soluble and membrane-bound DBH. Experiments in cultured bovine cells showed that labeled soluble and membrane-bound DBH had parallel turnover patterns and at all chase period, the distribution of radiolabeled DBH between the soluble contents and membranes was similar to the DBH activity distribution (50% soluble/50% membrane-bound). The above experiments showed that the soluble contents and membranes turnover in parallel and are consistent with nonreutilization of chromaffin granule membranes following exocytosis. Isolated retrograde perfused bovine adrenal glands were subjected to repetitive acetylcholine stimulation to induce exocytosis and then the dense and less-dense chromaffin granule fractions were isolated. Since both approaches gave results consistent with membrane nonreutilization, the authors conclude that once a chromaffin granule is involved in exocytosis, its membrane is not reutilized for the further synthesis, storage, and secretion of catecholamines

  8. Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

    International Nuclear Information System (INIS)

    In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 μM) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 μM veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 μM dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 μM) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated

  9. Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells.

    Science.gov (United States)

    Chan, Wing Hei; Gonsalvez, David G; Young, Heather M; Southard-Smith, E Michelle; Cane, Kylie N; Anderson, Colin R

    2016-02-01

    Adrenal medullary chromaffin cells and peripheral sympathetic neurons originate from a common sympathoadrenal (SA) progenitor cell. The timing and phenotypic changes that mark this lineage diversification are not fully understood. The present study investigated the expression patterns of phenotypic markers, and cell cycle dynamics, in the adrenal medulla and the neighboring suprarenal ganglion of embryonic mice. The noradrenergic marker, tyrosine hydroxylase (TH), was detected in both presumptive adrenal medulla and sympathetic ganglion cells, but with significantly stronger immunostaining in the former. There was intense cocaine and amphetamine-regulated transcript (CART) peptide immunostaining in most neuroblasts, whereas very few adrenal chromaffin cells showed detectable CART immunostaining. This phenotypic segregation appeared as early as E12.5, before anatomical segregation of the two cell types. Cell cycle dynamics were also examined. Initially, 88% of Sox10 positive (+) neural crest progenitors were proliferating at E10.5. Many SA progenitor cells withdrew from the cell cycle at E11.5 as they started to express TH. Whereas 70% of neuroblasts (TH+/CART+ cells) were back in the cell cycle at E12.5, only around 20% of chromaffin (CART negative) cells were in the cell cycle at E12.5 and subsequent days. Thus, chromaffin cell and neuroblast lineages showed differences in proliferative behavior from their earliest appearance. We conclude that the intensity of TH immunostaining and the expression of CART permit early discrimination of chromaffin cells and sympathetic neuroblasts, and that developing chromaffin cells exhibit significantly lower proliferative activity relative to sympathetic neuroblasts.

  10. Dynamin-2 regulates fusion pore expansion and quantal release through a mechanism that involves actin dynamics in neuroendocrine chromaffin cells.

    Directory of Open Access Journals (Sweden)

    Arlek M González-Jamett

    Full Text Available Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin's ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.

  11. The BAR Domain Protein PICK1 Controls Vesicle Number and Size in Adrenal Chromaffin Cells

    DEFF Research Database (Denmark)

    da Silva Pinheiro, Paulo César; Jansen, Anna M; de Wit, Heidi;

    2014-01-01

    Protein Interacting with C Kinase 1 (PICK1) is a Bin/Amphiphysin/Rvs (BAR) domain protein involved in AMPA receptor trafficking. Here, we identify a selective role for PICK1 in the biogenesis of large, dense core vesicles (LDCVs) in mouse chromaffin cells. PICK1 colocalized with syntaxin-6, a mar...

  12. F-actin cytoskeleton and the fate of organelles in chromaffin cells.

    Science.gov (United States)

    Villanueva, José; Gimenez-Molina, Yolanda; Viniegra, Salvador; Gutiérrez, Luis M

    2016-06-01

    In addition to playing a fundamental structural role, the F-actin cytoskeleton in neuroendocrine chromaffin cells has a prominent influence on governing the molecular mechanism and regulating the secretory process. Performing such roles, the F-actin network might be essential to first transport, and later locate the cellular organelles participating in the secretory cycle. Chromaffin granules are transported from the internal cytosolic regions to the cell periphery along microtubular and F-actin structures. Once in the cortical region, they are embedded in the F-actin network where these vesicles experience restrictions in motility. Similarly, mitochondria transport is affected by both microtubule and F-actin inhibitors and suffers increasing motion restrictions when they are located in the cortical region. Therefore, the F-actin cortex is a key factor in defining the existence of two populations of cortical and perinuclear granules and mitochondria which could be distinguished by their different location and mobility. Interestingly, other important organelles for controlling intracellular calcium levels, such as the endoplasmic reticulum network, present clear differences in distribution and much lower mobility than chromaffin vesicles and mitochondria. Nevertheless, both mitochondria and the endoplasmic reticulum appear to distribute in the proximity of secretory sites to fulfill a pivotal role, forming triads with calcium channels ensuring the fine tuning of the secretory response. This review presents the contributions that provide the basis for our current view regarding the influence that F-actin has on the distribution of organelles participating in the release of catecholamines in chromaffin cells, and summarizes this knowledge in simple models. In chromaffin cells, organelles such as granules and mitochondria distribute forming cortical and perinuclear populations whereas others like the ER present homogenous distributions. In the present review we discuss

  13. Cytosolic organelles shape calcium signals and exo-endocytotic responses of chromaffin cells.

    Science.gov (United States)

    García, Antonio G; Padín, Fernando; Fernández-Morales, José C; Maroto, Marcos; García-Sancho, Javier

    2012-01-01

    The concept of stimulus-secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca(2+)) entry and this generates a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers the exocytotic release of catecholamines. The control of the [Ca(2+)](c) signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca(2+) from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca(2+) extrusion mechanisms, such as the plasma membrane Ca(2+)-stimulated ATPase, and the Na(+)/Ca(2+) exchanger. Computation of the rates of Ca(2+) fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca(2+) transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca(2+)](c) transients occurring at different cell compartments, and generated by redistribution and release of Ca(2+) by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo-endocytotic activities with energy production and protein synthesis. PMID:22209033

  14. Regulation by L channels of Ca(2+)-evoked secretory responses in ouabain-treated chromaffin cells.

    Science.gov (United States)

    De Pascual, Ricardo; Colmena, Inés; Ruiz-Pascual, Lucía; Baraibar, Andrés Mateo; Egea, Javier; Gandía, Luis; García, Antonio G

    2016-10-01

    It is known that the sustained depolarisation of adrenal medullary bovine chromaffin cells (BCCs) with high K(+) concentrations produces an initial sharp catecholamine release that subsequently fades off in spite depolarisation persists. Here, we have recreated a sustained depolarisation condition of BCCs by treating them with the Na(+)/K(+) ATPase blocker ouabain; in doing so, we searched experimental conditions that permitted the development of a sustained long-term catecholamine release response that could be relevant during prolonged stress. BCCs were perifused with nominal 0Ca(2+) solution, and secretion responses were elicited by intermittent application of short 2Ca(2+) pulses (Krebs-HEPES containing 2 mM Ca(2+)). These pulses elicited a biphasic secretory pattern with an initial 30-min period with secretory responses of increasing amplitude and a second 30-min period with steady-state, non-inactivating responses. The initial phase was not due to gradual depolarisation neither to gradual increases of the cytosolic calcium transients ([Ca(2+)]c) elicited by 2Ca(2+) pulses in BBCs exposed to ouabain; both parameters increased soon after ouabain addition. Νifedipine blocked these responses, and FPL64176 potentiated them, suggesting that they were triggered by Ca(2+) entry through non-inactivating L-type calcium channels. This was corroborated by nifedipine-evoked blockade of the L-type Ca(2+) channel current and the [Ca(2+)]c transients elicited by 2Ca(2+) pulses. Furthermore, the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) blocker SEA0400 caused a mild inhibition followed by a large rebound increase of the steady-state secretory responses. We conclude that these two phases of secretion are mostly contributed by Ca(2+) entry through L calcium channels, with a minor contribution of Ca(2+) entry through the reverse mode of the NCX.

  15. Primary study on microencapsulated bovine adrena chromaffin cell xenotransplantation for chronic fermial pain%微囊化牛肾上腺嗜铬细胞脊髓蛛网膜下移植治疗慢性疼痛患者的初步观察

    Institute of Scientific and Technical Information of China (English)

    薛毅珑; 朱建华; 罗芸; 钟大光; 李雁凌; 何立敏; 李留树; 王捷; 王振福; 李新建; 张莉; 崔忻

    2000-01-01

    Objective To study the analgesic effect,the effective duration and the toxic-and side-effects of Alginate-Polylysine-Alginate (APA) microencapsulated bovine chromaffin cells (BCC) xenotransplanration on the terminal pain of 20 patients.Methods Sun's microencapsulation method was applied to encapsulate BCC with APA membrane and transplant the microencapsulated BCC(5~7)×106 into the subarachnoid space L3~5 of 20 patients.Results All 20 patients had pain relief rapidly after transplantation.Complete pain relief was shown in 14 cases,dramatic relief in 3 cases,medium relief in 1 case,slight relief in 2 cases.The rate for above medium relief was 90%.Without any immunosuppressants,3 patients remained pain free for over 200 days.Obvious toxic-and side-effects were not found.Conclusions Xenotransplantation of APA microencapsulated BCC into the spinal subarachnoid space of patients with cancer pain can promptly,significantly,safely produce analgesic effect for a long period.Microencapsulated BCC xenotransplantation may provide a unique and effective approach to the treatmerit of intractable chronic pain in human.%目的 观察海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊化牛肾上腺嗜铬细胞(BCC)异种移植对慢性顽固性疼痛患者的镇痛效应、作用持续时间及毒副作用.方法 用Sun氏微囊制作法将BCC包裹于APA微囊内,用常规腰穿法将5 ml微囊化BCC(5~7)×106悬液注入患者L3~5蛛网膜下.结果 20例中、重度慢性疼痛患者在1或2次注射后,疼痛迅速减轻,疼痛缓解率为90%.在未用任何免疫抑制剂的条件下,其中3例停用镇痛药时间超过200 d.结论 APA微囊化BCC异种移植于慢性疼痛患者脊髓蛛网膜下,可安全、迅速、长时间、有效地发挥镇痛作用.

  16. Membrane toxicity of abnormal prion protein in adrenal chromaffin cells of scrapie infected sheep.

    Directory of Open Access Journals (Sweden)

    Gillian McGovern

    Full Text Available Transmissible spongiform encephalopathies (TSEs or prion diseases are associated with accumulations of disease specific PrP (PrP(d in the central nervous system (CNS and often the lymphoreticular system (LRS. Accumulations have additionally been recorded in other tissues including the peripheral nervous system and adrenal gland. Here we investigate the effect of sheep scrapie on the morphology and the accumulation of PrP(d in the adrenal medulla of scrapie affected sheep using light and electron microscopy. Using immunogold electron microscopy, non-fibrillar forms of PrP(d were shown to accumulate mainly in association with chromaffin cells, occasional nerve endings and macrophages. PrP(d accumulation was associated with distinctive membrane changes of chromaffin cells including increased electron density, abnormal linearity and invaginations. Internalisation of PrP(d from the chromaffin cell plasma membrane occurred in association with granule recycling following hormone exocytosis. PrP(d accumulation and internalisation from membranes is similarly associated with perturbations of membrane structure and trafficking in CNS neurons and tingible body macrophages of the LRS. These data suggest that a major toxic effect of PrP(d is at the level of plasma membranes. However, the precise nature of PrP(d-membrane toxicity is tissue and cell specific suggesting that the normal protein may act as a multi-functional scaffolding molecule. We further suggest that the co-localisation of PrP(d with exocytic granules of the hormone trafficking system may provide an additional source of infectivity in blood.

  17. Regulation of Calcium Channels and Exocytosis in Mouse Adrenal Chromaffin Cells by Prostaglandin EP3 Receptors

    Science.gov (United States)

    Jewell, Mark L.; Breyer, Richard M.

    2011-01-01

    Prostaglandin (PG) E2 controls numerous physiological functions through a family of cognate G protein-coupled receptors (EP1–EP4). Targeting specific EP receptors might be therapeutically useful and reduce side effects associated with nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors that block prostanoid synthesis. Systemic immune challenge and inflammatory cytokines have been shown to increase expression of the synthetic enzymes for PGE2 in the adrenal gland. Catecholamines and other hormones, released from adrenal chromaffin cells in response to Ca2+ influx through voltage-gated Ca2+ channels, play central roles in homeostatic function and the coordinated stress response. However, long-term elevation of circulating catecholamines contributes to the pathogenesis of hypertension and heart failure. Here, we investigated the EP receptor(s) and cellular mechanisms by which PGE2 might modulate chromaffin cell function. PGE2 did not alter resting intracellular [Ca2+] or the peak amplitude of nicotinic acetylcholine receptor currents, but it did inhibit CaV2 voltage-gated Ca2+ channel currents (ICa). This inhibition was voltage-dependent and mediated by pertussis toxin-sensitive G proteins, consistent with a direct Gβγ subunit-mediated mechanism common to other Gi/o-coupled receptors. mRNA for all four EP receptors was detected, but using selective pharmacological tools and EP receptor knockout mice, we demonstrated that EP3 receptors mediate the inhibition of ICa. Finally, changes in membrane capacitance showed that Ca2+-dependent exocytosis was reduced in parallel with ICa. To our knowledge, this is the first study of EP receptor signaling in mouse chromaffin cells and identifies a molecular mechanism for paracrine regulation of neuroendocrine function by PGE2. PMID:21383044

  18. Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells.

    Science.gov (United States)

    Hao, Zhenhua; Wei, Lisi; Feng, Yaqin; Chen, Xiaowei; Du, Wen; Ma, Jing; Zhou, Zhuan; Chen, Liangyi; Li, Wei

    2015-04-01

    The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky-Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs.

  19. Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells.

    Science.gov (United States)

    Mahata, Sushil K; Zheng, Hong; Mahata, Sumana; Liu, Xuefei; Patel, Kaushik P

    2016-09-01

    One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic-adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition. PMID:27402067

  20. Neuronal localization of pituitary adenylate cyclase-activating polypeptide 38 in the adrenal medulla and growth-inhibitory effect on chromaffin cells

    DEFF Research Database (Denmark)

    Frödin, M; Hannibal, J; Wulff, B S;

    1995-01-01

    medulla showed PACAP38 immunoreactivity in a widely distributed network of delicate nerve fibers surrounding the chromaffin cells. In a primary culture system, PACAP38 inhibited growth factor-stimulated DNA synthesis by 90% in neonatal and adult rat chromaffin cells with half-maximal inhibition at 4 and 0...... cells, 100 nM PACAP38 and 1 microM corticosterone added together abolished proliferation completely (99.8% inhibition). Finally, PACAP38 increased cell survival but showed little neurite-promoting activity in the chromaffin cells. Our data suggest that neurally derived PACAP38, in conjunction...

  1. Transformation of adrenal medullary chromaffin cells increases asthmatic susceptibility in pups from allergen-sensitized rats

    Directory of Open Access Journals (Sweden)

    Feng Jun-Tao

    2012-11-01

    Full Text Available Abstract Background Studies have shown that epinephrine release is impaired in patients with asthma. The pregnancy of female rats (dams with asthma promotes in their pups the differentiation of adrenal medulla chromaffin cells (AMCCs into sympathetic neurons, mediated by nerve growth factor, which leads to a reduction in epinephrine secretion. However, the relatedness between the alteration of AMCCs and increased asthma susceptibility in such offspring has not been established. Methods In this study, we observed the effects of allergization via ovalbumin on rat pups born of asthmatic dams. Results Compared to the offspring of untreated controls, bronchial hyperreactivity and airway inflammation were more severe in the pups from sensitized (asthmatic dams. In pups exposed to nerve growth factor (NGF in utero these effects were aggravated further, but the effects were blocked in pups whose dams had been treated with anti-NGF. Furthermore, alterations in AMCC phenotype corresponded to the degree of bronchial hyperreactivity and lung lesions of the different treatment groups. Such AMCC alterations included degranulation of chromaffin granules, reduction of epinephrine and phenylethanolamine-n-methyl transferase, and elevation of NGF and peripherin levels. Conclusions Our results present evidence that asthma during the pregnancy of rat dams promotes asthma susceptibility in their offspring, and that the transformation of AMCCs to neurons induced by NGF plays an important role in this process.

  2. Rab3 proteins involved in vesicle biogenesis and priming in embryonic mouse chromaffin cells

    DEFF Research Database (Denmark)

    Schonn, Jean-Sébastien; van Weering, Jan R T; Mohrmann, Ralf;

    2010-01-01

    chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD(-/-) animals large dense core vesicles (LDCVs) are less abundant while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces...... the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD(-/-) cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher...... rate after an initial delay. Rescue experiments showed that short-term (4-6 hours) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV...

  3. A new diamond biosensor with integrated graphitic microchannels for detecting quantal exocytic events from chromaffin cells

    CERN Document Server

    Picollo, Federico; Vittone, Ettore; Pasquarelli, Alberto; Carbone, Emilio; Olivero, Paolo; Carabelli, Valentina

    2013-01-01

    The quantal release of catecholamines from neuroendocrine cells is a key mechanism which has been investigated with a broad range of materials and devices, among which carbon-based materials such as carbon fibers, diamond-like carbon, carbon nanotubes and nanocrystalline diamond. In the present work we demonstrate that a MeV-ion-microbeam lithographic technique can be successfully employed for the fabrication of an all-carbon miniaturized cellular bio-sensor based on graphitic micro-channels embedded in a single-crystal diamond matrix. The device was functionally characterized for the in vitro recording of quantal exocytic events from single chromaffin cells, with high sensitivity and signal-to-noise ratio, opening promising perspectives for the realization of monolithic all-carbon cellular biosensors.

  4. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

    Science.gov (United States)

    Suchard, S J; Lattanzio, F A; Rubin, R W; Pressman, B C

    1982-09-01

    The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation. PMID:7130269

  5. MiR-124 is differentially expressed in derivatives of the sympathoadrenal cell lineage and promotes neurite elongation in chromaffin cells.

    Science.gov (United States)

    Shtukmaster, Stella; Narasimhan, Priyanka; El Faitwri, Tehani; Stubbusch, Jutta; Ernsberger, Uwe; Rohrer, Hermann; Unsicker, Klaus; Huber, Katrin

    2016-08-01

    The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells. PMID:27094431

  6. Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

    Science.gov (United States)

    Ferrero, Rut; Torres, Magdalena

    2002-01-01

    Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

  7. Insulin-like growth factors act synergistically with basic fibroblast growth factor and nerve growth factor to promote chromaffin cell proliferation

    DEFF Research Database (Denmark)

    Frödin, M; Gammeltoft, S

    1994-01-01

    and immunocytochemical staining of cell nuclei. After 6 days in culture in the absence of growth factors, nuclear BrdUrd incorporation was detected in 30% of fetal chromaffin cells, 1.5% of neonatal cells, and 0.1% of adult cells. Addition of 10 nM IGF-I or IGF-II increased the fraction of BrdUrd-labeled nuclei to 50......% of fetal, 20% of neonatal, and 2% of adult chromaffin cells. The ED50 value of IGF-I- and IGF-II-stimulated BrdUrd labeling in neonatal chromaffin cells was 0.3 nM and 0.8 nM, respectively. In neonatal and adult chromaffin cells, addition of 1 nM bFGF or 2 nM NGF stimulated nuclear BrdUrd incorporation...... was further improved when bFGF or NGF was added together with IGF-II. In conclusion, we propose that IGF-I and IGF-II act in synergy with bFGF and NGF to stimulate proliferation and survival of chromaffin cells during neonatal growth and adult maintenance of the adrenal medulla. Our findings may have...

  8. Trichosporin-B-III, an alpha-aminoisobutyric acid-containing peptide, causes Ca(2+)-dependent catecholamine secretion from adrenal medullary chromaffin cells.

    Science.gov (United States)

    Tachikawa, E; Takahashi, S; Furumachi, K; Kashimoto, T; Iida, A; Nagaoka, Y; Fujita, T; Takaishi, Y

    1991-11-01

    We examined the effect of trichosporin-B-III, an alpha-aminoisobutyric acid-containing antibiotic peptide consisting of 19 amino acid residues and a phenylalaninol, on catecholamine secretion from cultured bovine adrenal chromaffin cells. Incubation of the cells with trichosporin-B-III (3-20 microM) caused an increase in the secretion of catecholamines. The secretion induced by trichosporin-B-III at low concentrations (3 and 5 microM) was completely dependent on external Ca2+, whereas that induced by higher concentrations (10 and 20 microM) was partly independent of Ca2+. Trichosporin-B-III at low concentration (5 microM) did not increase the release of lactate dehydrogenase, a marker enzyme of cytoplasm, from the cells. In contrast, the peptide at higher concentration (10 microM) increased the release of the enzyme. Trichosporin-B-III also caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to trichosporin-B-III concentration (3-10 microM). The time courses of the increases in secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration induced by trichosporin-B-III were also quite similar. Trichosporin-B-III-induced (at 5 microM) secretion was not affected by the elimination of Na+ from the incubation medium or by the addition of tetrodotoxin, a blocker of highly selective voltage-dependent Na+ channels, or hexamethonium, a blocker of nicotinic acetylcholine receptors. On the other hand, both diltiazem (2-200 microM) and nicardipine (1-200 microM), blockers of voltage-dependent Ca2+ channels, inhibited the secretion induced by trichosporin-B-III (5 microM) in a concentration-dependent manner. Trichosporin-B-III-induced (at 5 microM) secretion also was suppressed by the addition of Mn2+ (5 mM) to the medium. The diltiazem (20 microM) inhibition of trichosporin-B-III-induced (at 5 microM) secretion was reversed by

  9. Impact of Chromogranin A deficiency on catecholamine storage, catecholamine granule morphology and chromaffin cell energy metabolism in vivo.

    Science.gov (United States)

    Pasqua, Teresa; Mahata, Sumana; Bandyopadhyay, Gautam K; Biswas, Angshuman; Perkins, Guy A; Sinha-Hikim, Amiya P; Goldstein, David S; Eiden, Lee E; Mahata, Sushil K

    2016-03-01

    Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell. PMID:26572539

  10. Asthma pregnancy alters postnatal development of chromaffin cells in the rat adrenal medulla.

    Directory of Open Access Journals (Sweden)

    Xiu-Ming Wu

    Full Text Available BACKGROUND: Adrenal neuroendocrine plays an important role in asthma. The activity of the sympathoadrenal system could be altered by early life events. The effects of maternal asthma during pregnancy on the adrenal medulla of offspring remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: This study aims to explore the influence of maternal asthma during pregnancy on the development and function of adrenal medulla in offspring from postnatal day 3 (P3 to postnatal day 60 (P60. Asthmatic pregnant rats (AP, nerve growth factor (NGF-treated pregnant rats (NP and NGF antibody-treated pregnant rats (ANP were sensitized and challenged with ovalbumin (OVA; NP and ANP were treated with NGF and NGF antibody respectively. Offspring rats from the maternal group were divided into four groups: offspring from control pregnant rats (OCP, offspring from AP (OAP, offspring from NP (ONP, and offspring from ANP (OANP. The expressions of phenylethanolamine N-methyltransferase (PNMT protein in adrenal medulla were analyzed. The concentrations of epinephrine (EPI, corticosterone and NGF in serum were measured. Adrenal medulla chromaffin cells (AMCC were prone to differentiate into sympathetic nerve cells in OAP and ONP. Both EPI and PNMT were decreased in OAP from P3 to P14, and then reached normal level gradually from P30 to P60, which were lower from birth to adulthood in ONP. Corticosterone concentration increased significantly in OAP and ONP. CONCLUSION/SIGNIFICANCE: Asthma pregnancy may promote AMCC to differentiate into sympathetic neurons in offspring rats and inhibit the synthesis of EPI, resulting in dysfunction of bronchial relaxation.

  11. Intranuclear bundles of microfilaments and microtubules in chromaffin cells of the auricle of the heart of a lungfish, Protopterus aethiopicus.

    Science.gov (United States)

    Scheuermann, D W; Adriaensen, D; De Groodt-Lasseel, M H

    1988-01-01

    Intranuclear microtubular-microfilamentous rod-like inclusions were investigated in chromaffin cells of the auricle of the heart of lungfishes. In conventional electron microscopy, these inclusions reveal a wide variety in appearance, depending on their orientation to the plane of sectioning. Whereas originally they were merely interpreted as a bundle of microfilaments, application of a goniometer stage showed the rod- or spindle-shaped intranuclear inclusions to have a basic substructure of parallel arranged microtubules among microfilaments, which are clearly connected to chromatin granules, occasionally penetrating dense areas of chromatin. The chemical nature and biological significance of these structures, which so far remain enigmatic, are discussed. PMID:3227775

  12. Impaired desensitization of a human polymorphic α2B-adrenergic receptor variant enhances its sympatho-inhibitory activity in chromaffin cells

    Directory of Open Access Journals (Sweden)

    Lymperopoulos Anastasios

    2011-02-01

    Full Text Available Abstract Background α2-adrenergic receptors (ARs mediate many cellular actions of epinephrine and norepinephrine and inhibit their secretion from adrenal chromaffin cells. Like many other G-protein coupled receptors (GPCRs, they undergo agonist-dependent phopshorylation and desensitization by GPCR Kinases (GRKs, a phenomenon recently shown to play a major role in the sympathetic overdrive that accompanies and aggravates chronic heart failure. A deletion polymorphism in the human α2B-AR gene (Glu301-303 causes impaired agonist-promoted receptor phosphorylation and desensitization in heterologous cell lines. Given the importance of α2-ARs in regulation of catecholamine secretion from chromaffin cells, we sought to investigate, in the present study, the desensitization properties and the sympatho-inhibitory activity of this variant in a chromaffin cell line. For this purpose, we expressed this variant and its wild type counterpart in the well-established chromaffin cell line PC12, and performed receptor phosphorylation and desensitization studies, as well as in vitro catecholamine secretion assays. Results Both the agonist-induced phosphorylation and agonist-dependent desensitization of the human Glu301-303 deletion polymorphic α2B-AR are significantly impaired in PC12 cells, resulting in enhanced signaling to inhibition of cholinergic-induced catecholamine secretion in vitro. Conclusion This α2B-AR gene polymorphism (Glu301-303 deletion might confer better protection against conditions characterized and aggravated by sympathetic/catecholaminergic overstimulation in vivo.

  13. Microelectrode arrays of diamond-insulated graphitic channels for real time detection of exocytotic events from cultured chromaffin cells and slices of adrenal glands

    CERN Document Server

    Picollo, F; Bernardi, E; Marcantoni, A; Pasquarelli, A; Carbone, E; Olivero, P; Carabelli, V

    2016-01-01

    A microstructured graphitic 4x4 multielectrode array was embedded in a single crystal diamond substrate (4x4 {uG-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time effective fabrication of extended arrays with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20x3.5 um^2) separated by 200 um gaps. Taking advantage of the array geometry we addressed the following specific issues: i) detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, ii) resolve the waveform of different subsets of exocytotic events, iii) monitoring quantal secretory events from thin slices of ...

  14. Muted protein is involved in the targeting of CD63 to large dense-core vesicles of chromaffin cells.

    Science.gov (United States)

    Zhenhua, Hao; Wei, Li

    2016-08-01

    Large dense-core vesicles (LDCVs) are characterized as a class of lysosome-related organelles (LROs), which undergo regulated release and play important roles in development, metabolism and homeostasis. The Muted protein is a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), which functions in the biogenesis of lysosomes and LROs. CD63 is a membrane component of lysosomes and LROs. Whether and how CD63 is sorted into LDCVs is largely unknown. In this study, we aim to identify the localization of CD63 in chromaffin cells by colocalization, living cell imaging and cell fractionation. We found that a proportion of CD63-YFP colocalized with NPY-dsRed labeled LDCVs. By sucrose density gradient fractionation, a proportion of CD63 was found to be highly enriched in LDCVs fractions. The Muted mutant mouse is a model of Hermansky-Pudlak syndrome (HPS). We also found that the level of CD63 was significantly decreased in Muted-deficient adrenal glands, suggesting that the Muted protein is important for the steady-state level of CD63. Our results suggest that CD63 is a membrane component of LDCVs and the stability of CD63 is dependent on the Muted protein, which provides a clue to the pathogenesis of LRO defects in HPS. PMID:27531610

  15. The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.

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    Chih-Wei Chou

    Full Text Available BACKGROUND: While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio. Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a or matrix metalloproteinase 2 (mmp2, two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through

  16. Immunoreactive atrial natriuretic peptide and dopamine beta-hydroxylase in myocytes and chromaffin cells of the heart of the African lungfish, Protopterus aethiopicus.

    Science.gov (United States)

    Larsen, T H; Helle, K B; Saetersdal, T

    1994-07-01

    The heart of the African lungfish, Protopterus aethiopicus, was examined for immunoreactive atrial natriuretic peptide (ANP) and dopamine beta-hydroxylase (D beta H) as markers for hormone secreting myocytes and chromaffin cells, respectively. Specific antibodies raised against rat alpha-ANP and rat D beta H were used for immunofluorescence microscopy and immunogold electron microscopy. D beta H-immunoreactive cells were restricted to subendocardial areas of the atrium whereas ANP immunoreactivity occurred throughout both the atrial and the ventricular myocardium, showing particularly strong staining intensity in the atrial myocytes. The granular ANP immunostaining in the atrial myocytes was frequently accumulated in the sarcoplasm. In the ventricular myocytes ANP immunoreactivity occurred as scattered granular staining throughout the sarcoplasm. ANP and D beta H immunofluorescence staining coincided with the presence of immunoreactive specific granules and secretory vesicles in the cardiac myocytes and chromaffin cells, respectively, as revealed by electron microscopy. The number of ANP-containing specific granules was generally high in the atrial myocytes, and they were frequently observed in clusters in subsarcolemmal areas. Granular frequency was considerably lower and the mean granular diameter was smaller (0.142 +/- 0.045 micron versus 0.213 +/- 0.049 micron) in the ventricular than in the atrial myocytes. The present results indicate that ANP and D beta H are phylogenetically highly conserved proteins from the dipnoi to the rat. The large amounts of ANP and of specific granules are consistent with an endocrine myocardium in the Protopterus heart. The presence of D beta H and secretory vesicles in the subendocardial chromaffin cells of the atrium suggests a local production of catecholamines from dopamine in the heart of this dipnoan. PMID:7926645

  17. Butanol isomers exert distinct effects on voltage-gated calcium channel currents and thus catecholamine secretion in adrenal chromaffin cells.

    Directory of Open Access Journals (Sweden)

    Sarah McDavid

    Full Text Available Butanol (C4H10OH has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I(Ca is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I(Ca. We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.

  18. Butanol isomers exert distinct effects on voltage-gated calcium channel currents and thus catecholamine secretion in adrenal chromaffin cells.

    Science.gov (United States)

    McDavid, Sarah; Bauer, Mary Beth; Brindley, Rebecca L; Jewell, Mark L; Currie, Kevin P M

    2014-01-01

    Butanol (C4H10OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I(Ca)) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I(Ca). We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.

  19. Sustained Exocytosis after Action Potential-Like Stimulation at Low Frequencies in Mouse Chromaffin Cells Depends on a Dynamin-Dependent Fast Endocytotic Process

    Science.gov (United States)

    Moya-Díaz, José; Álvarez, Yanina D.; Montenegro, Mauricio; Bayonés, Lucas; Belingheri, Ana V.; González-Jamett, Arlek M.; Cárdenas, Ana M.; Marengo, Fernando D.

    2016-01-01

    Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 ± 0.11 s, fast enough to maintain synchronous exocytosis at 0.2–0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 ± 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies. PMID:27507935

  20. SUSTAINED EXOCYTOSIS AFTER ACTION POTENTIAL-LIKE STIMULATION AT LOW FREQUENCIES IN MOUSE CHROMAFFIN CELLS DEPENDS ON A DYNAMIN-DEPENDENT FAST ENDOCYTOTIC PROCESS

    Directory of Open Access Journals (Sweden)

    José Moya-Díaz

    2016-07-01

    Full Text Available Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls. The exocytosis triggered by APls (ETAP represents a fraction (40% of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 +/- 0.11 s, fast enough to maintain synchronous exocytosis at 0.2-0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ=0.53±0.01 s. In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz. Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies.

  1. A Post-Docking Role of Synaptotagmin 1-C2B Domain Bottom Residues R398/399 in Mouse Chromaffin Cells

    DEFF Research Database (Denmark)

    Kedar, Girish H; Munch, Anders S; van Weering, Jan R T;

    2015-01-01

    , Syt1-R398/399Q (RQ), in syt1 null mutant cells. Ultrastructural morphometry revealed that Syt1-RQ fully restored the docking defect observed previously in syt1 null mutant cells, similar to wild type Syt1 (Syt1-wt). Small unilamellar lipid vesicles (SUVs) that contained the v-SNARE Synaptobrevin2...... and Syt1-R398/399Q also docked to t-SNARE-containing giant vesicles (GUVs), similar to Syt1-wt. However, unlike Syt1-wt, Syt1-RQ-induced docking was strictly PI(4,5)P2-dependent. Unlike docking, neither synchronized secretion in chromaffin cells nor Ca2+-triggered SUV–GUV fusion was restored by the Syt1...

  2. Bovine endometrial stromal cells display osteogenic properties

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    Cavirani Sandro

    2008-12-01

    Full Text Available Abstract The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.

  3. Microelectrode Arrays of Diamond-Insulated Graphitic Channels for Real-Time Detection of Exocytotic Events from Cultured Chromaffin Cells and Slices of Adrenal Glands.

    Science.gov (United States)

    Picollo, Federico; Battiato, Alfio; Bernardi, Ettore; Marcantoni, Andrea; Pasquarelli, Alberto; Carbone, Emilio; Olivero, Paolo; Carabelli, Valentina

    2016-08-01

    A microstructured graphitic 4 × 4 multielectrode array was embedded in a single-crystal diamond substrate (4 × 4 μG-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time-effective fabrication of extended arrays with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20 × 3.5 μm(2)) separated by 200 μm gaps. Taking advantage of the array geometry we addressed the following specific issues: (i) detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, (ii) resolve the waveform of different subsets of exocytotic events, and (iii) monitoring quantal secretory events from thin slices of the adrenal gland. The frequency of spontaneous release was low (0.12 and 0.3 Hz, respectively, for adrenal slices and cultured cells) and increased up to 0.9 Hz after stimulation with 30 mM KCl in cultured cells. The spike amplitude as well as rise and decay time were comparable with those measured by carbon fiber microelectrodes and allowed to identify three different subsets of secretory events associated with "full fusion" events, "kiss-and-run" and "kiss-and-stay" exocytosis, confirming that the device has adequate sensitivity and time resolution for real-time recordings. The device offers the significant advantage of shortening the time to collect data by allowing simultaneous recordings from cell populations either in primary cell cultures or in intact tissues. PMID:27376596

  4. Spatial distribution and temporal evolution of DRONPA-fused SNAP25 clusters in adrenal chromaffin cells

    DEFF Research Database (Denmark)

    Antoku, Yasuko; Dedecker, Peter; da Silva Pinheiro, Paulo César;

    2015-01-01

    Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized......, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the DRONPA-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes....

  5. NUTRIENTS AND EPIGENETICS IN BOVINE CELLS

    Science.gov (United States)

    This is a chapter for a book titled “Livestock Epigenetics” edited by Dr. Hasan Khatib and published by Wiley-Blackwell. This chapter is focused on the research development in our laboratory in the area of interaction of nutrients and genomic phonotype in bovine cells. Briefly, the Research on nutri...

  6. The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

    NARCIS (Netherlands)

    Lammers, A.; Vorstenbosch, van C.J.; Erkens, J.H.F.; Smith, H.E.

    2001-01-01

    We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover. we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other importan

  7. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

    Science.gov (United States)

    The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and wer...

  8. Detection of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea Viruses in the Nasal Epithelial Cells by the Direct Immunofluorescence Technique

    OpenAIRE

    Silim, A.; Elazhary, M. A. S. Y.

    1983-01-01

    Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 µL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell culture...

  9. Cell-free translation of bovine viral diarrhea virus RNA.

    OpenAIRE

    Purchio, A F; Larson, R.; Torborg, L L; Collett, M S

    1984-01-01

    Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to...

  10. Generation of bovine transgenics using somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Stice Steven L

    2003-11-01

    Full Text Available Abstract The ability to produce transgenic animals through the introduction of exogenous DNA has existed for many years. However, past methods available to generate transgenic animals, such as pronuclear microinjection or the use of embryonic stem cells, have either been inefficient or not available in all animals, bovine included. More recently somatic cell nuclear transfer has provided a method to create transgenic animals that overcomes many deficiencies present in other methods. This review summarizes the benefits of using somatic cell nuclear transfer to create bovine transgenics as well as the possible opportunities this method creates for the future.

  11. Differentiation of Bovine Spermatogonial Stem Cells into Osteoblasts

    OpenAIRE

    Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

    2011-01-01

    Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 da...

  12. Propagation of bovine spermatogonial stem cells in vitro

    NARCIS (Netherlands)

    Aponte, P.M.; Soda, T.; Teerds, K.J.; Mizrak, S.C.; Kant, van de H.J.; Rooij, de D.G.

    2008-01-01

    The access to sufficient numbers of spermatogonial stem cells (SSC) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behaviour of bovine type A spermatogonia, a cell population that incl

  13. Proteomic analysis of bovine blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille Linnert; Grøndahl, Marie Louise; Beck, Hans Christian;

    2014-01-01

    Abstract The understanding of the early mammalian development is a prerequisite for the advancement of in vitro fertilization and improvement of derivation and culturing of embryonic stem cells. While, whole genome transcriptomic analysis on bovine blastocysts has identified genes active in early...... development, little information is available about the protein complement of early embryos. Modern, sensitive proteomic technology (nano HPLC tandem mass spectrometry) allowed us to describe the proteome of the scarce blastocoel fluid and cell material of expanded bovine blastocysts isolated...... by micromanipulation. From two independent replicates, 23 proteins were identified in the blastocoel fluid while 803 proteins were identified in the remaining cell material. The proteins were grouped into categories according to their gene ontology (GO) terms by which proteins involved in cell differentiation, cell...

  14. Multilineage Potential Research of Bovine Amniotic Fluid Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yuhua Gao

    2014-02-01

    Full Text Available The use of amnion and amniotic fluid (AF are abundant sources of mesenchymal stem cells (MSCs that can be harvested at low cost and do not pose ethical conflicts. In human and veterinary research, stem cells derived from these tissues are promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. This work aimed to obtain and characterize bovine amniotic fluid mesenchymal stem cells (AFMSC. The bovine AF from the amniotic cavity of pregnant gilts in the early stages of gestation (3- and 4-m-old bovine embryos was collected. AFMSCs exhibit a fibroblastic-like morphology only starting from the fourth passage, being heterogeneous during the primary culture. Immunofluorescence results showed that AFMSCs were positive for β-integrin, CD44, CD73 and CD166, but negative for CD34, CD45. Meanwhile, AFMSCs expressed ES cell markers, such as Oct4, and when appropriately induced, are capable of differentiating into ectodermal and mesodermal lineages. This study reinforces the emerging importance of these cells as ideal tools in veterinary medicine; future studies aimed at a deeper evaluation of their immunological properties will allow a better understanding of their role in cellular therapy.

  15. Effects of Visible Light on Cultured Bovine Trabecular Cells

    Institute of Scientific and Technical Information of China (English)

    姜发纲; 郝风芹; 魏厚仁; 许德胜

    2004-01-01

    To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1. 12 mW/cm2 , the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.

  16. Bovine mammary stem cells: new perspective for dairy science.

    Science.gov (United States)

    Martignani, E; Cravero, D; Miretti, S; Accornero, P; Baratta, M

    2014-01-01

    Mammary stem cells provide opportunities for the cyclic remodelling of the bovine mammary gland. Therefore, understanding the character and regulation of mammary stem cells is important for increasing animal health and productivity. The exciting possibility that stem cell expansion can influence milk production is currently being investigated by several researchers. In fact, appropriate regulation of mammary stem cells could hopefully benefit milk yield, persistency of lactation, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and regulate the function of bovine mammary stem cells. However, research on mammary stem cells requires tissue biopsies, which represents a limitation for the management of animal welfare. Interestingly, different studies recently reported the identification of putative mammary stem cells in human breast milk. The possible identification of primitive cell types within cow's milk may provide a non-invasive source of relevant mammary cells for a wide range of applications. In this review, we have summarized the main achievements in this field for dairy cow science and described the interesting perspectives open to manipulate milk persistency during lactation and to cope with oxidative stress during the transition period by regulating mammary stem cells.

  17. Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Tong, W.

    1997-10-08

    Microscale separation and detection methods for real-time monitoring of dynamic cellular processes (e.g., secretion) by capillary electrophoresis (CE) and microscopic imaging were developed. Ultraviolet laser-induced native fluorescence (LINF) provides simple, sensitive and direct detection of neurotransmitters and proteins without any derivatization. An on-column CE-LINF protocol for quantification of the release from single cell was demonstrated. Quantitative measurements of both the amount of insulin released from and the amount remaining in the cell ({beta}TC3) were achieved simultaneously. Secretion of catecholamines (norepinephrine (NE) and epinephrine (E)) from individual bovine adrenal chromaffin cells was determined using the on-column CE-LINF. Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved by LINF imaging microscopy with high temporal and spatial resolution. The secretion of serotonin from individual leech Retzius neurons was directly characterized by LINF microscopy with high spatial resolution.

  18. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Crane, M.St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K. (Merck Sharp and Dohme Research Labs., Rahway, NJ (USA))

    1984-06-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G/sub 2/ phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed.

  19. Local IL2 and IL12 treatment of Bovine Ocular Squamous Cell Carcinoma (BOSCC) and Bovine Vulval Papilloma and Carcinoma Complex (BVPCC) in Cattle in Zimbabwe

    NARCIS (Netherlands)

    Stewart, R.J.E.

    2007-01-01

    Effect of local IL-2 application on bovine cancer In tropical countries there is an increased prevalence of two important cancers in cattle: Bovine Ocular Squamous Cell Carcinoma (BOSCC) and Bovine Vulval Papilloma Carcinoma Complex (BVPCC). Both cancers are associated with increased annual hours of

  20. Morphological aspects of chromaffin tissue: the differential fixation of adrenaline and noradrenaline.

    OpenAIRE

    Kobayashi, S; Coupland, R. E.

    1993-01-01

    The morphological aspects of chromaffin tissue are reviewed, based mainly on our studies on the mouse adrenal gland. Particular attention was focused on the differential fixation of adrenaline and noradrenaline, and on the uptake and storage of [3H]dopa, [3H]dopamine and related substances in the adrenaline-storing (A) and noradrenaline-storing (NA) cells. Scanning electron microscopy combined with the NaOH-maceration method was useful for demonstrating the 3-dimensional organisation of nerve...

  1. Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells.

    Science.gov (United States)

    Hermosilla, C; Barbisch, B; Heise, A; Kowalik, S; Zahner, H

    2002-04-01

    Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.

  2. Identification of major cell types in paraffin sections of bovine tissues

    OpenAIRE

    Pessa-Morikawa Tiina; Ekman Anna; Niku Mikael; Iivanainen Antti

    2006-01-01

    Abstract Background Identification of cell types in bovine tissue sections is complicated by the limited availability of anti-bovine antibodies, and by antigen retrieval treatments required for formalin-fixed tissue samples. We have evaluated an antibody and lectin panel for identifying major cell types in paraffin-embedded bovine tissue sections, and report optimized pretreatments for these markers. Results We selected 31 useful antibodies and lectins which can be used to identify cell types...

  3. 人嗜铬细胞微囊化处理对大鼠异种移植的免疫隔离作用%Immunoisolated effect of xenografts of microencapsulated human chromaffin cells in rats

    Institute of Scientific and Technical Information of China (English)

    陈冀衡; 杨茂元; 李继勇; 林献忠; 毕好生

    2008-01-01

    组和微囊化人嗜铬细胞移植组均低于人嗜铬细胞移植组,差异有显著性意义(t=8.544~21.64,P < 0.01).②大鼠眼前房和足胝部组织形态:人嗜铬细胞移植组大鼠的眼前房内和足胝部可见大量淋巴细胞和中性粒细胞浸润.空微囊移植组和微囊化人嗜铬细胞移植组大鼠眼前房和足胝部仅见少量淋巴细胞和中性粒细胞.结论:海藻酸钠-多聚赖氨酸-海藻酸钠微囊化所产生的良好生物相容性及其机械稳定性,使之有效地发挥了免疫排斥隔离作用.%BACKGROUND: Based on previous technique prepared for encapsulating living cells with alginate-polysine- alginate (APA) microcapsules, it has been confirmed that microencapsulated chromaffin cells have good analgesic effects. The immunoisolated effects of such microcapsule materials need to be evaluated. OBJECTIVE: This study aimed to investigate the immunological rejections of APA microencapsulated chromaffin cells transplanted into rat anterior chamber of eyes and tendon of feet, and to evaluate the immunoisolated effect of microencapsulation.DESIGN: A randomized controlled animal experiment. SETTING: Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Forty-eight female SD rats, with the age of 3 months, were provided by the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology. The protocol was carried out in accordance with ethical guidelines for the use and care of animals. Alginate and polylysine used in the experiment were the products of Sigma Company, USA. Microcapsule generator was gifted by Germany. METHODS: This study was performed at the Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology from September 2002 to September 2003. Suprarenal medulla was taken from 6 healthy adult cadavers of brain death. After isolated

  4. Effect of dietary bovine colostrum on the responses of immune cells to stimulation with bacterial lipopolysaccharide.

    Science.gov (United States)

    Xu, Mei Ling; Kim, Hyoung Jin; Kim, Hong-Jin

    2014-04-01

    Previous studies have revealed that ingestion of bovine colostrum is effective in preventing pathogens from invading through the gastrointestinal tract (GI) and modulating the mucosal immunity of the GI tract, indicating that its effect is principally local. Thus it is unclear if ingestion of bovine colostrum can affect the systemic immune system. In this study, we investigated the effect of taking bovine colostrum (vs phosphate-buffered saline) for 14 days on the behavior of the immune cells of mice. Isolated splenocytes, which are pivotal cells of systemic immunity, were then stimulated with Escherichia coli lipopolysaccharide. Bovine colostrum significantly reduced NK cell and monocyte activities and lymphoproliferaltive responses to LPS stimulation. Thus dietary bovine colostrum renders immune cells less responsive to LPS stimulation. Dietary bovine colostrum thus affects the systemic immune system and may have anti-inflammatory actions. PMID:24234910

  5. Effects of putrescine, cadaverine, spermine, spermidine and beta-phenylethylamine on cultured bovine mammary epithelial cells

    DEFF Research Database (Denmark)

    Fusi, Eleonora; Baldi, Antonella; Cheli, Federica;

    2008-01-01

    A bovine mammary epithelial cell line (BME-UV1) and three-dimensional collagen primary bovine organoids were used to evaluate the effects of cadaverine, putrescine, spermine, spermicline and beta-phenylethylamine on mammary epithelial cells. Each biogenic amine was diluted in several concentratio...

  6. Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

    Science.gov (United States)

    Malaver-Ortega, Luis F; Sumer, Huseyin; Jain, Kanika; Verma, Paul J

    2016-02-01

    Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro--two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation. PMID:26660942

  7. Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine.

    OpenAIRE

    Scherman, D; Jaudon, P; Henry, J.P.

    1983-01-01

    [2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-11b-H-benzo [a]-quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane of purified bovine chromaffin granules. Specific binding was characterized by Kd and Bmax values of 3.1 nM and 62 pmol/mg of membrane protein, respectively. It was reversible, with association and dissociation rate constants of 0.22 x 10(6) M-1 s-1 and 1.8 x 10(-3) s-1, respectively. Binding sites were present...

  8. Enrichment for Repopulating Cells and Identification of Differentiation Markers in the Bovine Mammary Gland.

    Science.gov (United States)

    Rauner, Gat; Barash, Itamar

    2016-06-01

    Elucidating cell hierarchy in the mammary gland is fundamental for understanding the mechanisms governing its normal development and malignant transformation. There is relatively little information on cell hierarchy in the bovine mammary gland, despite its agricultural potential and relevance to breast cancer research. Challenges in bovine-to-mouse xenotransplantation and difficulties obtaining bovine-compatible antibodies hinder the study of mammary stem-cell dynamics in this species. In-vitro indications of distinct bovine mammary epithelial cell populations, sorted according to CD24 and CD49f expression, have been provided. Here, we successfully transplanted these bovine populations into the cleared fat pads of immunocompromised mice, providing in-vivo evidence for the multipotency and self-renewal capabilities of cells that are at the top of the cell hierarchy (termed mammary repopulating units). Additional outgrowths from transplantation, composed exclusively of myoepithelial cells, were indicative of unipotent basal stem cells or committed progenitors. Sorting luminal cells according to E-cadherin revealed three distinct populations: luminal progenitors, and early- and late-differentiating cells. Finally, miR-200c expression was negatively correlated with differentiation levels in both the luminal and basal branches of the bovine mammary cell hierarchy. Together, these experiments provide further evidence for the presence of a regenerative entity in the bovine mammary gland and for the multistage differentiation process within the luminal lineage. PMID:26615610

  9. Ethanolamine metabolism in cultured bovine aortic endothelial cells

    International Nuclear Information System (INIS)

    The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells. Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine

  10. Cell Infectivity in Relation to Bovine Leukemia Virus gp51 and p24 in Bovine Milk Exosomes

    OpenAIRE

    Yamada, Tetsuya; Shigemura, Hiroaki; ISHIGURO, Naotaka; Inoshima, Yasuo

    2013-01-01

    Exosomes are small membranous microvesicles (40–100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exo...

  11. Cell Infectivity in Relation to Bovine Leukemia Virus gp51 and p24 in Bovine Milk Exosomes

    OpenAIRE

    Tetsuya Yamada; Hiroaki Shigemura; Naotaka Ishiguro; Yasuo Inoshima

    2013-01-01

    Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exo...

  12. Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

    Institute of Scientific and Technical Information of China (English)

    AN Li-long; YANG Qi; XIAO Mei; FENG Xiu-Liang; YANG Chun-rong; LEI An-min; GAO Zhi-min; DOU Zhong-ying; QIU Huai

    2002-01-01

    Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary marine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS ,0.1μmol/L Na2SeO3, 0. 1mmol/L β-mercaptoethanol, 1 000ng/ml LIF,10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine), then, we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage marine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0. 125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification, karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.

  13. Transcriptomic microarray analysis of BoMac cells after infection with bovine foamy virus

    NARCIS (Netherlands)

    Rola-Luszczak, M.; Materniak, M.; Pluta, A.; Hulst, M.M.; Kuz'mak, J.

    2014-01-01

    Bovine foamy virus (BFV) infections are highly prevalent among cattle worldwide. However, relatively little is known about the impact of this virus on the host immune system. In our study, we focused on a bovine macrophage cell line (BoMac) and examined changes in the BoMac transcriptome after in vi

  14. Cytotoxicity of bovine and porcine collagen membranes in mononuclear cells.

    Science.gov (United States)

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Carneiro, Karine Fernandes; Souza, Maria Aparecida de; Magalhães, Denildo

    2012-01-01

    This study compared the cytotoxicity and the release of nitric oxide induced by collagen membranes in human mononuclear cells. Peripheral blood was collected from each patient and the separation of mononuclear cells was performed by Ficoll. Then, 2x10(5) cells were plated in 48-well culture plates under the membranes in triplicate. The polystyrene surface was used as negative control. Cell viability was assessed by measuring mitochondrial activity (MTT) at 4, 12 and 24 h, with dosage levels of nitrite by the Griess method for the same periods. Data had non-normal distribution and were analyzed by the Kruskal-Wallis test (p<0.05). Statistically significant differences (p<0.05) were observed between the membranes and the control in the experimental period, although there was a significant reduction in viability over time (p<0.01). At 4 and 12 h, the porcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (p<0.01), and this difference was maintained at 24 h (p<0.05). This in vitro study showed that the porcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time. PMID:22460313

  15. Dietary bovine lactoferrin increases intestinal cell proliferation in neonatal piglets.

    Science.gov (United States)

    Reznikov, Elizabeth A; Comstock, Sarah S; Yi, Cuiyi; Contractor, Nikhat; Donovan, Sharon M

    2014-09-01

    Lactoferrin is a bioactive milk protein that stimulates cell proliferation in vitro; however, limited in vivo evidence exists to allow lactoferrin to be incorporated into infant formula. Herein, the effect of dietary bovine lactoferrin (bLF) on neonatal intestinal growth and maturation was investigated guided by the hypothesis that bLF would increase cellular proliferation leading to functional differences in neonatal piglets. Colostrum-deprived piglets were fed formula containing 0.4 [control (Ctrl)], 1.0 (LF1), or 3.6 (LF3) g bLF/L for the first 7 or 14 d of life. To provide passive immunity, sow serum was provided orally during the first 36 h of life. Intestinal cell proliferation, histomorphology, mucosal DNA concentration, enzyme activity, gene expression, and fecal bLF content were measured. Intestinal enzyme activity, DNA concentration, and villus length were unaffected by bLF. However, crypt proliferation was 60% greater in LF1- and LF3-fed piglets than in Ctrl piglets, and crypt depth and area were 20% greater in LF3-fed piglets than in Ctrl piglets. Crypt cells from LF3-fed piglets had 3-fold higher β-catenin mRNA expression than did crypt cells from Ctrl piglets. Last, feces of piglets fed bLF contained intact bLF, suggesting that some bLF was resistant to digestion and could potentially affect intestinal proliferation through direct interaction with intestinal epithelial cells. This study is the first to our knowledge to show that dietary bLF stimulates crypt cell proliferation in vivo. The increased β-catenin expression indicates that Wnt signaling may in part mediate the stimulatory effect of bLF on intestinal cell proliferation. PMID:25056692

  16. Zfp423 promotes adipogenic differentiation of bovine stromal vascular cells.

    Directory of Open Access Journals (Sweden)

    Yan Huang

    Full Text Available Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423 as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05 in high adipogenic cells, while transforming growth factor (TGF-β was higher (156.1±48.7%, P<0.05 in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ and CCAAT/enhancer binding protein α (C/EBPα were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05 in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular

  17. Bovine mammary stem cells: Cell biology meets production agriculture

    Science.gov (United States)

    Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue ...

  18. Differentiation in neuroblastoma: diffusion-limited hypoxia induces neuro-endocrine secretory protein 55 and other markers of a chromaffin phenotype.

    Directory of Open Access Journals (Sweden)

    Fredrik Hedborg

    Full Text Available BACKGROUND: Neuroblastoma is a childhood malignancy of sympathetic embryonal origin. A high potential for differentiation is a hallmark of neuroblastoma cells. We have previously presented data to suggest that in situ differentiation in tumors frequently proceeds along the chromaffin lineage and that decreased oxygen (hypoxia plays a role in this. Here we explore the utility of Neuro-Endocrine Secretory Protein 55 (NESP55, a novel member of the chromogranin family, as a marker for this process. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analyses and in situ hybridizations were performed on human fetal tissues, mouse xenografts of human neuroblastoma cell lines, and on specimens of human neuroblastoma/ganglioneuroma. Effects of anaerobic exposure on gene expression by cultured neuroblastoma cells was analyzed with quantitative real-time PCR. Fetal sympathetic nervous system expression of NESP55 was shown to be specific for chromaffin cell types. In experimental and clinical neuroblastoma NESP55 immunoreactivity was specific for regions of chronic hypoxia. NESP55 expression also correlated strikingly with morphological evidence of differentiation and with other chromaffin-specific patterns of gene expression, including IGF2 and HIF2α. Anaerobic culture of five neuroblastoma cell lines resulted in an 18.9-fold mean up-regulation of NESP55. CONCLUSIONS/SIGNIFICANCE: The data confirms that chronic tumor hypoxia is a key microenvironmental factor for neuroblastoma cell differentiation, causing induction of chromaffin features and NESP55 provides a reliable marker for this neuronal to neuroendocrine transition. The hypoxia-induced phenotype is the predominant form of differentiation in stroma-poor tumors, while in stroma-rich tumors the chromaffin phenotype coexists with ganglion cell-like differentiation. The findings provide new insights into the biological diversity which is a striking feature of this group of tumors.

  19. Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells.

    Science.gov (United States)

    Tebaldi, Giulia; Jacca, Sarah; Montanini, Barbara; Capra, Emanuele; Rosamilia, Alfonso; Sala, Arianna; Stella, Alessandra; Castiglioni, Bianca; Ottonello, Simone; Donofrio, Gaetano

    2016-07-01

    Viral infections can cause genital tract disorders (including abortion) in cows, and bovine herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation. PMID:27281703

  20. Interaction of bovine peripheral blood polymorphonuclear cells and Leptospira species; innate responses in the natural bovine reservoir host.

    Directory of Open Access Journals (Sweden)

    Jennifer H Wilson-Welder

    2016-07-01

    Full Text Available Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and can also be reservoir hosts of other Leptospira species such as L. kirschneri, and L. interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murine neutrophils have shown activation of neutrophil extracellular trap or NET formation, and upregulation of inflammatory mediators by neutrophils in the presence of Leptospira. Humans, companion animals and most widely studied models of Leptospirosis are of acute infection, hallmarked by systemic inflammatory response, neutrophilia and septicemia. In contrast, cattle exhibit chronic infection with few outward clinical signs aside from reproductive failure. Taking into consideration that there is host species variation in innate immunity, especially in pathogen recognition and response, the interaction of bovine peripheral blood polymorphonuclear cells (PMNs and several Leptospira strains was evaluated. Studies including bovine-adapted strains, human pathogen strains, a saprophyte and inactivated organisms. Incubation of PMNs with Leptospira did induce slight activation of neutrophil NETs, greater than unstimulated cells but less than the quantity from E. coli P4 stimulated PMNs. Very low but significant from non-stimulated, levels of reactive oxygen peroxides were produced in the presence of all Leptospira strains and E. coli P4. Similarly, significant levels of reactive nitrogen intermediaries (NO2 was produced from PMNs when incubated with the Leptospira strains and greater quantities in the presence of E. coli P4. PMNs incubated with Leptospira induced RNA transcripts of IL-1β, MIP-1α, and TNF-α, with greater amounts induced by live organisms when compared to heat-inactivated leptospires. Transcript for inflammatory cytokine IL-8 was also induced, at similar levels regardless of Leptospira strain or viability. However, incubation of

  1. THE CHARACTERISTICS OF ENDOGENOUS OUABAIN SECRETIONFROM CULTURED BOVINE ADRENOCORTICAL CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To compare the characteristics of endogenous ouabain(EO) secretion with the other adrenocortical hormones and determine the effects of angiotensin Ⅰ (Ang Ⅰ ), and adrenocorticotrophin(ACTH) on the secretion of EO. Methods EO was measured by radioimmunoassay from primary cultured bovine adrenocotical cells (BAC). Results ①Ouabain was determined in the media of cultured BAC. Both EO and aldosterone secretion were decreased from the outer to inner layer of the cultured adrenal cortex, and the responses to Ang Ⅰ and ACTH were higher than that in the mid layer (P <0. 05) and inner layer (P <0. 01). Cortisol secretion was activated by Ang Ⅱ or ACTH was significantly higher in the mid layer and in the inner layer than that in the outer layer. ②The time-course experiment showed that the gradually rising amounts of aldosterone and cortisol could be determined dur ing the continuous incubation to 48h with or without Ang Ⅰ or ACTH. However, EO did not increase continuously af ter 24h of incubation in the basal secreting situation and after 12h of incubation in the stimulating situation by Ang Ⅱ or ACTH. ③There were obvious drops in aldosterone and cortisol secretion from 3rd day during a 21 day-period cell culture, but the peak secretion of ouabain was in 7th day. Conclusion It suggests that the secretory mechanism might be different between EO and aldosterone or cortisol. Also, Ang Ⅱ and ACTH might be involved in the regulation of EO secretion.

  2. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

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    Ricardo Chaves Vilela

    2013-02-01

    Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

  3. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

    Science.gov (United States)

    Chinchar, V G; Floyd, A D; Chinchar, G D; Taylor, M W

    1979-02-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

  4. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells.

    OpenAIRE

    Oprian, D D; Molday, R S; Kaufman, R. J.; Khorana, H G

    1987-01-01

    We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in...

  5. Somatic cell bovine cloning: Effect of donor cell and recipients

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Adult somatic cell nuclear transfer was conducted by using cultured ear fibroblast cells obtained from a Holstein female cow (GN) and a Galoway herd bull (GLV). The percentages of reconstructed eggs developed into blastocysts were similar in GN (23.98%, 123 of 513) and in GLV groups (29.55%, 138 of 467). However, the rate of reconstructed female (GN) embryos developed into term was higher than that of male (GLV) (8.02% and 1.82%, respectively). Three kinds of cows, Luxi Yellow cows, Holstein heifers and Holstein cows with normal reproductive records were used as recipients. When the reconstructed embryos from GN were transferred, there was no difference in the pregnancy rate among three kinds of recipients, but the abortion rate of Luxi Yellow cows was significantly higher (85.71%) than in the other two groups (14.29% and 0%, respectively; P < 0.05). And the percentages of newborn calves in transferred embryos were significantly different between Luxi Yellow cows and Holstein breed (1.54%, 10.39% and 20.0%, respectively, P < 0.05). However, when reconstructed embryos from GLV were transferred, there was no difference among three kinds of recipients in the pregnancy rate, the abortion rate and the delivery rate.

  6. Generation of a persistently infected MDBK cell line with natural bovine spongiform encephalopathy (BSE).

    Science.gov (United States)

    Tark, Dongseob; Kim, Hyojin; Neale, Michael H; Kim, Minjeong; Sohn, Hyunjoo; Lee, Yoonhee; Cho, Insoo; Joo, Yiseok; Windl, Otto

    2015-01-01

    Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrPBSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrPBSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.

  7. Generation of a persistently infected MDBK cell line with natural bovine spongiform encephalopathy (BSE.

    Directory of Open Access Journals (Sweden)

    Dongseob Tark

    Full Text Available Bovine spongiform encephalopathy (BSE is a zoonotic transmissible spongiform encephalopathy (TSE thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD. Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrPBSE and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrPBSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.

  8. Regulatory Effect of Dexamethasone on Aquaporin-1 Expression in Cultured Bovine Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    XIONG Xinchun; MIAO Juan; XI Zulian; ZHANG Haijiang; HAN Bo; HU Yizhen

    2005-01-01

    To evaluate the effect of dexamethasone on the expression of aquaporin-1 (AQP-1) in cultured bovine trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and reproduced to the third and the fourth generation, then treated with dexamethasone at the concentrations of 5, 25, 50, 250 μg/L respectively for 7 days. Immunohistochemical technique-supervision method was employed to measure, and image analysis system to analyze the expression of AQP-1 in normal cultured bovine trabecular meshwork cells and those treated with dexamethasone.In normal bovine trabecular meshwork cells, the grayscale of AQP-1 positive staining was 167.94±1.18, while it was 168.92±0.91, 176.72±1.80, 180.64±1.31, 185.64±1.58 in cells treated with 5, 25, 50, 250 tg/L concentrations of dexamethasone. When the concentration of dexamethasone was higher than 25 μg/L, the expression of AQP-1 was significantly inhibited (P<0.05).The regulation of AQP-1 expression by dexamethasone in cultured bovine trabecular meshwork cells in vitro may be one of causes that retard the aqueous outflow in glucocorticoid induced glaucoma.

  9. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro.

    Science.gov (United States)

    Sipka, Anja; Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-10-01

    Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46(+)/CD3(-)) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis. PMID:27638120

  10. Alternative splicing regulated by butyrate in bovine epithelial cells.

    Directory of Open Access Journals (Sweden)

    Sitao Wu

    Full Text Available As a signaling molecule and an inhibitor of histone deacetylases (HDACs, butyrate exerts its impact on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. This study examined the effect of butyrate on alternative splicing in bovine epithelial cells using RNA-seq technology. Junction reads account for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT and control (CT groups. 201,326 potential splicing junctions detected were supported by ≥ 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG while ~3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon skipping events, supported by a minimum of 3 junction reads, were detected. At least 7 genes, their mRNA expression significantly affected by butyrate, also had exon skipping events differentially regulated by butyrate. Furthermore, COL5A3, which was induced 310-fold by butyrate (FDR <0.001 at the gene level, had a significantly higher number of junction reads mapped to Exon#8 (Donor and Exon#11 (Acceptor in BT. This event had the potential to result in the formation of a COL5A3 mRNA isoform with 2 of the 69 exons missing. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate physically binds to and inhibits all zinc-dependent HDACs except HDAC6 and HDAC10. Our results provided evidence that butyrate also regulated deacetylase activities of classical HDACs via its transcriptional control. Moreover, thirteen gene fusion events differentially affected by butyrate were identified. Our results provided a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of the biological effects of butyrate and other HDAC

  11. Measuring bovine gamma delta T cell function at the site of Mycobacterium bovis infection

    Science.gov (United States)

    The causative agent of tuberculosis (TB) in cattle is Mycobacterium bovis. The characteristic lesions of bovine TB are well-organized pulmonary granulomas. Gamma delta T cells are a unique subset of nonconventional T cells that play major roles in both the innate and adaptive arms of the immune sys...

  12. Targeting exogenous GDNF gene to the bovine somatic cell beta-casein locus for the production of transgenic bovine animals.

    Science.gov (United States)

    Zhang, X M; Luo, F H; Ding, H M; Li, B; Zhang, J J; Wu, Y J

    2015-01-01

    Considerable attention is currently being directed toward methods for producing recombinant human proteins in the mammary glands of genetically modified transgenic livestock. However, the expression of inserted genes in transgenic animals is variable and often very low because of the randomness of the site of transgene integration. One possible strategy to avoid the expression problem associated with random integration is to use site-specific integration by targeting integration to a high expression locus and, thereby, to improve expression of the transferred gene. In the present study, we focused on glial cell line-derived neurotrophic factor (GDNF), a novel type of neurotrophic factor first cloned in 1993. Research has shown that GDNF may have potential applications in the treatment of Parkinson's disease and other diseases of the central nervous system since it acts as a protective factor for central dopaminergic neurons. Here, we constructed a gene targeting vector to knock-in the human GDNF gene at the bovine beta-casein gene locus as a first step to producing transgenic animals with a high level of expression of human GDNF protein in their mammary glands. Bovine fetal fibroblast cells were transfected with linearized pNRTCNbG by electroporation. Three cell clones were identified with successful targeting to the beta-casein locus; and were confirmed using both polymerase chain reaction analysis and sequencing. Gene-targeted cells were used as nuclear donors; a total of 161 embryos were reconstructed, 23 of which developed to the blastocyst stage. These blastocysts were transferred to 8 recipient cows, but no offspring were obtained. PMID:26634460

  13. Immunohistochemistry studies on bovine squamous cell carcinoma morphological characterization of epidermal cell proliferation and differentiation markers and characterization of cytokeratins

    OpenAIRE

    Vala, Helena; Fondevila, D.; Carvalho, T.; Pinto, C.; Peleteiro, C.; Pinho, M.; Ferrer, L

    2001-01-01

    Bovine Ocular Squamous Cell Carcinoma (OSCC) is a general designation for a group of primary neoplasias of keratinocytes arising from ocular tissues, especially the lids and particularly the third eye lid. OSCC has been diagnosed all over the world with high prevalence, being the most common bovine tumour and the one causing the most significant economic losses (Hamir & Parry, 1980; Dennis et al., 1985, Heeney & Valli, 1985; Wilcock, 1993). In Portugal, the frequency of these tumours...

  14. Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells.

    Science.gov (United States)

    Han, Zhao-Yu; Mu, Tian; Yang, Zhen

    2015-01-01

    The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.

  15. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

    NARCIS (Netherlands)

    Sipka, Anja; Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-01-01

    Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3) in bovine mammary gland tissue after an intramammary challenge with Escheri

  16. Effects of interferon-tau and steroids on cytochrome P450 activity in bovine endometrial epithelial cells

    Science.gov (United States)

    The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon-t (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were tr...

  17. Research on Growth Behavior of Embryos for Bovine and Murine on Primary Murine Embryos Fibroblast Cell Feeder Layer

    Institute of Scientific and Technical Information of China (English)

    AN Li-long; XIAO Mei; FENG Xiu-Liang; DOU Zhong-ying; QIU Huai; YANG Qi; LEI An-min; YANG Chun-rong; GAO Zhi-min

    2002-01-01

    The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the mixture-like colonies. Compared with Murine ICM, the bovine ICM grew more fast. So, the bovine ICM was passaged at first after a culture of approximately 5 - 6 days in vitro, but murine ICM was passaged at first after an attachment of 3 - 4 days on PMEF feeder layer. The mixture colonies of bovine ICM differentiated very early, while the others differentiated very late. Most ICM-like mass of Bovine grew in a defined spot, but bovine ICMs like stream and ICMs like net proliferated fast and dispersed quickly. We found that the single blastomeres derived from late bovine morula and late murine morula formed sub-blastophere; moreover, the bovine ICM cell would differentiate rapidly if the trophoblast was removed.

  18. Screen of Bovine Mammary Gland Epithelial Cell Specifcity Promotor

    Institute of Scientific and Technical Information of China (English)

    Liu Xiao-fei; Li Qing-zhang; Qiu You-wen; Gao Xue-jun

    2012-01-01

    Three lactoproteins (α-Sl-casein, β-lactoglobulin, and β-casein) promotors were cloned, sequenced and compared relative luciferase expression. The results showed that the promotor activity of bovine α-S1-casein gene was the best, and would be used to produce pharmaceutically and medically important proteins in the mammary gland of transgenic animals and also for the construction of an inducible eukaryotic expression vector.

  19. Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus.

    Science.gov (United States)

    Kempster, S; Collins, M E; Brownlie, J

    2002-03-01

    The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established.

  20. Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells

    Directory of Open Access Journals (Sweden)

    Schilling William P

    2001-02-01

    Full Text Available Abstract Background Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i via activation of Ca2+-permeable, non-selective cation channels (CaNSC. Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells. Results Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da, YO-PRO-1 (Mw 375 Da, and POPO-3 (Mw 715 Da showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation. Conclusions These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.

  1. Characterization of bovine A20 gene: Expression mediated by NF-κB pathway in MDBK cells infected with bovine viral diarrhea virus-1.

    Science.gov (United States)

    Fredericksen, Fernanda; Villalba, Melina; Olavarría, Víctor H

    2016-05-01

    Cytokine production for immunological process is tightly regulated at the transcriptional and posttranscriptional levels. The NF-κB signaling pathway maintains immune homeostasis in the cell through the participation of molecules such as A20 (TNFAIP3), which is a key regulatory factor in the immune response, hematopoietic differentiation, and immunomodulation. Although A20 has been identified in mammals, and despite recent efforts to identify A20 members in other higher vertebrates, relatively little is known about the composition of this regulator in other classes of vertebrates, particularly for bovines. In this study, the genetic context of bovine A20 was explored and compared against homologous genes in the human, mouse, chicken, dog, and zebrafish chromosomes. Through in silico analysis, several regions of interest were found conserved between even phylogenetically distant species. Additionally, a protein-deduced sequence of bovine A20 evidenced many conserved domains in humans and mice. Furthermore, all potential amino acid residues implicated in the active site of A20 were conserved. Finally, bovine A20 mRNA expression as mediated by the bovine viral diarrhea virus and poly (I:C) was evaluated. These analyses evidenced a strong fold increase in A20 expression following virus exposure, a phenomenon blocked by a pharmacological NF-κB inhibitor (BAY 117085). Interestingly, A20 mRNA had a half-life of only 32min, likely due to adenylate- and uridylate-rich elements in the 3'-untranslated region. Collectively, these data identify bovine A20 as a regulator of immune marker expression. Finally, this is the first report to find the bovine viral diarrhea virus modulating bovine A20 activation through the NF-κB pathway. PMID:26809100

  2. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    C Lin

    Full Text Available Our previous studies showed that bovine respiratory syncytial virus (BRSV followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2 epithelial cells with H. somni concentrated culture supernatant (CCS stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2 and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

  3. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM cells in cattle naturally infected with bovine tuberculosis.

    Directory of Open Access Journals (Sweden)

    Adam O Whelan

    Full Text Available Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2. Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+IL-2(+TNF-α(+ and IFN-γ(+ TNF-α(+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hiCD45RO(+CD62L(lo T-effector memory (T(EM phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

  4. Apoptosis is induced in bovine satellite muscle cells after removal of available oxygen

    OpenAIRE

    Andersen, Petter Vejle

    2013-01-01

    Abstract Post-mortem tenderisation of meat is a complex process, of which all the details are far from understood. Cell death by apoptosis is recently proposed as a novel mechanism in this process. The main aim of this study was to investigate if bovine satellite muscle cells, cultivated in vitro, would induce apoptosis when oxygen was removed from the incubation medium. Satellite muscle cells was seeded out in Entactin-Collagen IV-Laminin (ECL) coated culture wells and allowed to diffe...

  5. Enhanced adipogenic differentiation of bovine bone marrow-derived mesenchymal stem cells

    Science.gov (United States)

    Until now, the isolation and characterization of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimize the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three 6-month-old cattle and used for p...

  6. Virus-host interactions in persistently FMDV-infected cells derived from bovine pharynx

    Science.gov (United States)

    Foot-and-mouth disease virus (FMDV) produces a disease in cattle characterized by vesicular lesions and a persistent infection with asymptomatic low-level production of virus. Here we describe the establishment of a persistently infected primary cell culture derived from bovine pharynx tissue (PBPT)...

  7. INSULIN INDUCES NITRIC OXIDE PRODUCTION IN BOVINE AORTIC ENDOTHELIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    李慧丽; 黄定九

    2000-01-01

    ffeStun6. ObjeCtif Dens cette dtude nons avons ewilue l' effet de l' insuline sur ba Proliferation cellulaire, liberationd' oxide nitrique et l' expression gdrktique de synthase d' oxide nitrique dens la cellule endotheliale aortique bovine. methIn mitogdthe est evalude per la ndthae M7T. In Production de NO dans ie alga en culture est determine per la reactionGness. In technique quantitative RT/PCR est utility pour quantifier ie niveau de sWthase d' oxide nitrique mRNA dens la cellule endotheliale aortique...

  8. Gamma Interferon Production by Bovine γδ T Cells following Stimulation with Mycobacterial Mycolylarabinogalactan Peptidoglycan

    Science.gov (United States)

    Vesosky, B.; Turner, O. C.; Turner, J.; Orme, I. M.

    2004-01-01

    A large percentage of lymphocytes in the blood of cattle express the γδ T-cell receptor, but specific functions for these cells have not yet been clearly defined. There is evidence, however, that human, murine, and bovine γδ T cells have a role in the immune response to mycobacteria. This study investigated the ability of bovine γδ T cells to expand and produce gamma interferon (IFN-γ) in response to stimulation with mycobacterial products. Bovine γδ T cells, isolated from the peripheral blood of healthy cattle, expanded following in vitro stimulation with live mycobacteria, mycobacterial crude cell wall extract, and Mycobacterium bovis culture filtrate proteins. In addition, purified γδ T cells, cocultured with purified monocytes and interleukin-2, consistently produced significant amounts of IFN-γ in response to mycobacterial cell wall. The IFN-γ-inducing component of the cell wall was further identified as a proteolytically resistant, non-sodium dodecyl sulfate-soluble component of the mycolylarabinogalactan peptidoglycan. PMID:15271921

  9. Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes

    OpenAIRE

    Lívia Maria Mendonça Augusto; Diego Pinheiro Aguiar; Danielle Cabral Bonfim; Amanda dos Santos Cavalcanti; Priscila Ladeira Casado; Maria Eugênia Leite Duarte

    2016-01-01

    ABSTRACT OBJECTIVE: This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. METHODS: Bovine tendons were used for preparation of the extract and were stored at -80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. RESULTS: The data showed that mesenchymal stromal cells from bone...

  10. In vitro replication activity of bovine viral diarrhea virus in an epithelial cell line and in bovine peripheral blood mononuclear cells.

    Science.gov (United States)

    Turin, Lauretta; Lucchini, Barbara; Bronzo, Valerio; Luzzago, Camilla

    2012-11-01

    The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.

  11. Bovine CD2-/NKp46+ cells are fully functional natural killer cells with a high activation status

    Science.gov (United States)

    Boysen, Preben; Olsen, Ingrid; Berg, Ingvild; Kulberg, Siri; Johansen, Grethe M; Storset, Anne K

    2006-01-01

    Background Natural killer (NK) cells in the cow have been elusive due to the lack of specific NK cell markers, and various criteria including a CD3-/CD2+ phenotype have been used to identify such cells. The recent characterization of the NK-specific NKp46 receptor has allowed a more precise definition of bovine NK cells. NK cells are known as a heterogeneous cell group, and we here report the first functional study of bovine NK cell subsets, based on the expression of CD2. Results Bovine CD2- NK cells, a minor subset in blood, proliferated more rapidly in the presence of IL-2, dominating the cultures after a few days. Grown separately with IL-2, CD2- and CD2+ NK cell subsets did not change CD2 expression for at least two weeks. In blood, CD2- NK cells showed a higher expression of CD44 and CD25, consistent with a high activation status. A higher proportion of CD2- NK cells had intracellular interferon-gamma in the cytoplasm in response to IL-2 and IL-12 stimulation, and the CD2- subset secreted more interferon-gamma when cultured separately. Cytotoxic capacity was similar in both subsets, and both carried transcripts for the NK cell receptors KIR, CD16, CD94 and KLRJ. Ligation by one out of two tested anti-CD2 monoclonal antibodies could trigger interferon-gamma production from NK cells, but neither of them could alter cytotoxicity. Conclusion These results provide evidence that bovine CD2- as well as CD2+ cells of the NKp46+ phenotype are fully functional NK cells, the CD2- subset showing signs of being more activated in the circulation. PMID:16643649

  12. Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity.

    Science.gov (United States)

    Wisnoskey, Brian J; Estacion, Mark; Schilling, William P

    2004-08-01

    The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca(2+) overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca(2+)-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba(2+) not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca(2+)-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba(2+). Taken together, these results suggest that intracellular high-affinity Ca(2+)-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise. PMID:15044153

  13. Characterization of putative receptors specific for quercetin on bovine aortic smooth-muscle cells

    International Nuclear Information System (INIS)

    The authors have reported that tobacco glycoprotein (TGP), rutin-bovine serum albumin conjugates (R-BSA), quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells (SMC). To investigate whether there are binding sites or receptors for these polyphenol-containing molecules on SMC, the authors have synthesized 125I-labeled rutin-bovine serum albumin ([125I]R-BSA) of high specific activity (20 Ci/mmol). SMC were isolated from a bovine thoracic aorta and maintained in Eagle's minimum essential medium with 10% calf serum in culture. These SMC at early subpassages were suspended (3-5 x 107 cells/ml) in phosphate-buffered saline and incubated with [125I]R-BSA (10 pmol) in the presence or absence of 200-fold unlabeled R-BSA, TGP, BSA, rutin, quercetin or related polyphenols, and catecholamines. Binding of [125I]R-BSA to SMC was found to be reproducible and the radioligand was displaced by R-BSA, and also by TGP, rutin, quercetin, and chlorogenic acid, but not by BSA, ellagic acid, naringin, hesperetin, dopamine, epinephrine, or isoproterenol. The binding was saturable, reversible, and pH-dependent. These results demonstrate the presence of specific binding sites for quercetinon arterial SMC

  14. Characterization of putative receptors specific for quercetin on bovine aortic smooth-muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, S.C.; Becker, C.G.

    1986-03-01

    The authors have reported that tobacco glycoprotein (TGP), rutin-bovine serum albumin conjugates (R-BSA), quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells (SMC). To investigate whether there are binding sites or receptors for these polyphenol-containing molecules on SMC, the authors have synthesized /sup 125/I-labeled rutin-bovine serum albumin ((/sup 125/I)R-BSA) of high specific activity (20 Ci/mmol). SMC were isolated from a bovine thoracic aorta and maintained in Eagle's minimum essential medium with 10% calf serum in culture. These SMC at early subpassages were suspended (3-5 x 10/sup 7/ cells/ml) in phosphate-buffered saline and incubated with (/sup 125/I)R-BSA (10 pmol) in the presence or absence of 200-fold unlabeled R-BSA, TGP, BSA, rutin, quercetin or related polyphenols, and catecholamines. Binding of (/sup 125/I)R-BSA to SMC was found to be reproducible and the radioligand was displaced by R-BSA, and also by TGP, rutin, quercetin, and chlorogenic acid, but not by BSA, ellagic acid, naringin, hesperetin, dopamine, epinephrine, or isoproterenol. The binding was saturable, reversible, and pH-dependent. These results demonstrate the presence of specific binding sites for quercetinon arterial SMC.

  15. Evaluation of bovine thymic function by measurement of signal joint T-cell receptor excision circles.

    Science.gov (United States)

    Hisazumi, Rinnosuke; Kayumi, Miya; Zhang, Weidong; Kikukawa, Ryuji; Nasu, Tetuo; Yasuda, Masahiro

    2016-01-01

    A signal joint T-cell receptor excision circle (sjTREC) is a circular DNA produced by T-cell receptor α gene rearrangement in the thymus. Measurements of sjTREC values have been used to evaluate thymic function. We recently established a quantitative PCR (QPCR) assay of bovine sjTREC. In the present study, we used this QPCR assay to measure the sjTREC value in bovine peripheral blood mononuclear cells and we then evaluated the relationships between sjTREC values and peripheral blood T-cell number, growth stage, gender, and meteorological season. The sjTREC value was highest at the neonatal stage, and its value subsequently decreased with age. On the other hand, the peripheral T-cell number increased with age. The sjTREC value in calves up to 50-days old was significantly higher for males than for females, suggesting that thymic function might differ by gender. In addition, the sjTREC value and the peripheral T-cell number were significantly higher in calves in the summer season than in calves in the winter season. These data suggest that bovine thymic function is highly variable and varies according to the growth stage, gender, and environmental factors such as air temperature or the UV index.

  16. Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes☆

    Science.gov (United States)

    Augusto, Lívia Maria Mendonça; Aguiar, Diego Pinheiro; Bonfim, Danielle Cabral; dos Santos Cavalcanti, Amanda; Casado, Priscila Ladeira; Duarte, Maria Eugênia Leite

    2016-01-01

    Objective This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. Methods Bovine tendons were used for preparation of the extract and were stored at −80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. Results The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. Conclusion Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes. PMID:26962503

  17. 309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

    DEFF Research Database (Denmark)

    Jensen, P L; Groendahl, M L; Beck, Helle;

    2012-01-01

    Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...... such as Parkinson's disease, diabetes, and spinal cord injury. One of today's challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine...... the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1-8nL per blastocyst) were isolated by micromanipulation and analysed by nano...

  18. Embryonic stem-like cells derived from in vitro produced bovine blastocysts

    Directory of Open Access Journals (Sweden)

    Erika Regina Leal de Freitas

    2011-06-01

    Full Text Available The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like cells from the inner cell mass (ICM of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35% ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF. Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

  19. Theileria parva: effects of irradiation on a culture of parasitized bovine lymphoid cells. [Gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Irvin, A.D.; Brown, C.G.D.; Stagg, D.A.

    1975-01-01

    Aliquots of a culture of Theileria parva-infected bovine lymphoid cells were irradiated at 0, 300, 600, 900, and 1200 rads. The short-term effects of irradiation were evaluated on examination of Giemsa-stained smears and on autoradiography of cells labeled with (/sup 3/H)thymidine. Irradiation inhibited cell division but parasite division did not appear to be inhibited and macroschizont nuclear particles increased in number, frequently to several hundred per schizont. There was no evidence of an increased percentage switch from macro- to microschizont. Apparently viable cells were still present in all cultures 4 days after irradiation.

  20. A bovine cell line that can be infected by natural sheep scrapie prions.

    Directory of Open Access Journals (Sweden)

    Anja M Oelschlegel

    Full Text Available Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice. We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  1. A bovine cell line that can be infected by natural sheep scrapie prions.

    Science.gov (United States)

    Oelschlegel, Anja M; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schatzl, Herman; Schaetzl, Hermann; Groschup, Martin H

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  2. Distribution of S-100 positive dendritic cells in bovine pharynx,tonsils, and retropharyngeal lymph nodes

    Institute of Scientific and Technical Information of China (English)

    Jiaxin WANG; Haixia BIAN; Wei SHI; Zhanjun LU

    2008-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells. However, the distribution of bovine DCs in the pharynx, tonsil, and retropharyngeal lymph nodes has not yet been documented. To address this issue, immunohistochemistry was conducted using S-100 pro-tein as a marker for DCs. It was observed that S-100 positive Langerhans cells (LCs) were primarily found in the basal layer of the pharyngeal epithelium. Some DCs were found in the outer layer of the epithelium and their dendrites extended out towards the epithelial surface. In the tonsil, S-100 positive DCs were found either in follicular germinal centers or in the T-cell areas. It is worth noting that the S-100 positive DCs were not only distributed in the cortex, but also in the medulla of bovine retropharyngeal lymph nodes. The distribution patterns of bovine DCs in the pharynx, tonsil, and retropharyngeal lymph nodes have an important implica-tion for our understanding of the interaction between pathogens and host.

  3. Muscarinic Receptor Agonists Protect Cultured Bovine Trabecular Meshwork Cells against Apoptosis Induced by Dexamethasone

    Institute of Scientific and Technical Information of China (English)

    Yajuan Gu; Shujun Zeng; PengXin Qiu; Yuping Wu; Dawei Peng; Guangmei Yan1

    2004-01-01

    Purpose:To study whether muscarinic receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone.Methods:The third to fifth passags of bovine trabecular meshwork cells were grown to confluence and incubated for 1~14 days in growth media with dexamethasone or pretreatment of pilocarpine or carbachol.The cultures were evaluated for apoptosis by phase-contrast microscopy, fluorescence microscopy, DNA laddering and flow cytometric analysis.Results :Dexamethasone (0.24~0.96 mmol·L-1) induced apoptosis of trabecular meshwork cells in a dose and time-dependent manner. Before 0.48 mmol·L-1 dexamethasone-treatment, 1.84 mmol· L-1 of pilocarpine or 2.74 mmol· L-1 of carbachol added could significantly reduce apoptotic percentage. Conclusion: Muscarinie receptor agonists can protect cultured bovine trabecular meshwork cells against apoptosis induced by dexamethasone. Eye Science 2004;20:42-47.

  4. Bovine colostrum modulates immune activation cascades in human peripheral blood mononuclear cells in vitro

    DEFF Research Database (Denmark)

    Jenny, Marcel; Pedersen, Ninfa R; Hidayat, Budi J;

    2010-01-01

    Bovine colostrum (BC) is the thick yellow fluid a lactating cow Oyes to a suckling calf during its first days of life to support the growth of the calf and prevent gastrointestinal infections until the calf has synthesized its own active immune defense system. BC contains a complex system of immune...... factors and has a long history of use in traditional medicine. In an approach to evaluate the effects of bovine colostrum (BC) on the T-cell/macrophage interplay, we investigated and compared the capacity of BC containing low and high amounts of lactose and lactoferrin to modulate tryptophan degradation...... and neopterin formation in unstimulated and mitogen-stimulated human peripheral blood mononuclear cells (PBMC). The present study shows significant immunomodulatory effects of these BC preparations in human PBMC, either by enhancing or suppressing the occurrence of a Th-1 type immune response. The amount...

  5. Characterization of T cell epitopes in bovine α-lactalbumin

    NARCIS (Netherlands)

    Meulenbroek, Laura A P M; den Hartog Jager, Constance F; Lebens, Ans F M; Knulst, André C; Bruijnzeel-Koomen, Carla A F M; Garssen, Johan; Knippels, Léon M J; van Hoffen, Els

    2014-01-01

    BACKGROUND: Recent studies have indicated that peptides containing T cell epitopes may be used for immunotherapy. While for several cow's milk allergens the T cell epitopes have been described, the T cell epitopes in the major allergen α-lactalbumin (α-LAC) are unknown. Therefore, the aim of this st

  6. Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

    Institute of Scientific and Technical Information of China (English)

    Yuping Feng; Jiao Wang; Shixin Ling; Zhuo Li; Mingsheng Li; Qiongyi Li; Zongren Ma; Sijiu Yu

    2014-01-01

    The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells fol-lowing induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined speciifc neu-ronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sec-tioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

  7. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    OpenAIRE

    Page Grier P; Kasinathan Poothappillai; Wang Zhongde; Rodriguez-Osorio Nelida; Robl James M; Memili Erdogan

    2009-01-01

    Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clo...

  8. Transcriptional Reprogramming of Gene Expression in Bovine Somatic Cell Chromatin Transfer Embryos

    OpenAIRE

    Rodriguez-Osorio, N.; Wang, Zhongde; Page, G. P.; Robl, J M; Memili, E.

    2009-01-01

    Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from ...

  9. Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

    OpenAIRE

    Bezek, D M; Baker, J. C.; Kaneene, J B

    1988-01-01

    A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood...

  10. Adenosine-5'-triphosphate release by Mannheimia haemolytica, lipopolysaccharide, and interleukin-1 stimulated bovine pulmonary epithelial cells.

    Science.gov (United States)

    Craddick, Michael; Patel, Rakhi; Lower, Amanda; Highlander, Sarah; Ackermann, Mark; McClenahan, David

    2012-09-15

    Mannheimia haemolytica, one of the agents associated with bovine respiratory disease complex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5'-triphosphate (ATP). Therefore, we wanted to determine if ATP levels were elevated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non-treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addition, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1. PMID:22771196

  11. Detection and analysis of bovine foamy virus infection by an indicator cell line

    Institute of Scientific and Technical Information of China (English)

    Zhe MA; Wen-tao QIAO; Cheng-hao XUAN; Jin-hui XIE; Qi-min CHEN; Yun-qi GENG

    2007-01-01

    Aim: To determine the infectivity and replication strategy of bovine foamy virus (BFV) in different cultured cells using the BFV indicator cell line (BICL) system. Methods: BFV infection was induced by the co-culture method or the transient transfection of the infectious BFV plasmid [Pcmv (cytomegalovirus) - BFV] clone. The infectivity of BFV was monitored by the percentage of green fluorescent protein-positive cells in the BICL. The effect of reverse transcriptase inhibitor zidovudine (AZT) on BFV replication was also evaluated in the BICL. Results-The titer of BFV in fetal bovine lung cells was 4-5-folds more than that in either 293T or HeLa (Cells from Henrietta lacks) cells using the co-culture method, and in the meantime was significantly higher than that produced by the infectious clone Pcmv-BFV in the same cells. AZT had only a minor effect on viral titers when added to cells prior to the virus infection. In contrast, viral titers reduced sharply to the level of the negative control when the virus was produced from cells in the presence of AZT. Conclusions: BICL can be used for the titration of the BFV viral infection in non-cytopathic condition. In addition, we provide important evi-dence to show that reverse transcription is essential for BFV replication at a late step of viral infection.

  12. Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

    Directory of Open Access Journals (Sweden)

    R. Bertazolli-Filho

    2007-10-01

    Full Text Available The trabecular meshwork (TM is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

  13. Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

    OpenAIRE

    Shaokun Zhang; Zaoxia Liu; Guanfang Su; Hong Wu

    2016-01-01

    The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human prima...

  14. Ultrastructural Aspects of the Prenatal Bovine Ovary Differentiation with a Special Focus on the Interstitial Cells.

    Science.gov (United States)

    Kenngott, R A-M; Scholz, W; Sinowatz, F

    2016-10-01

    The aim of this investigation was to study the ultrastructural features during the development of fetal bovine ovaries (crown rump length ranging from 11.4 to 94.0 cm). An interesting observation was the occurrence of big elongated cells containing a variety of electron dense granules and light homogenous vacuoles/bodies. They were located between the stroma cells surrounding the germ cell cord ends, adjacent to the first formed primordial follicles, typically situated near blood vessels. ER alpha and ER beta receptor positive cells could be detected in the same regions by means of immunohistochemistry. Intercellular bridges linked the germ cells nests oogonia. Germ cell cords consisted of centrally located, large, pale oogonia, surrounded by elongated somatic cells with very long cytoplasm extensions. Primordial follicles with flat pale follicular cells could be observed on the inner end of the cords. Extrusions of the outer nuclear membrane could often been recognised in voluminous oocytes.

  15. Bovine Mammary Epithelial Cell Lineages and Parenchymal Development

    Science.gov (United States)

    Mammary development proceeds from an aggregation of cells in the ventral ectoderm to the establishment of an elaborate tree of alveoli, ducts, and cisternae. However, despite abundant data on endocrine regulation of ruminant mammary growth, we know comparatively little about cell lineages, express...

  16. A Study of Toxic Effect of Mitomycin C on Cultured Bovine Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    Fagang Jiang; Houren Wei; Yuanshu Lu; Ying Zhang; Yuanqing Zhou

    2000-01-01

    Purpose: To explore the toxicity of Mitomycin C (MMC) on trabecular meshwork cells.Methods: Bovine trabecular meshwork cells were cultured in vitro and exposed to MMC of different concentrations. The cellular morphology, ultrastructure, mortality and phagocytosis was studied with light microscopy, transmission electron microscopy and methods of Wright's stain, etc.Results: It was found that the toxic effect of MMC on the cells was in a dose-dependent mode. 1 × 10-2 and 1 × 10-3mg/ml of MMC caused a large part of cells dead, 1 × 10-4 and 1 × 10-5mg/ml of the drug had remarkable killing effect on the cells. 1 × 10-6mg/ml of MMC had still a mild toxicity, while 1 × 10-7 mg/ml of MMC had not any influence on cellular morphology, mortality, and phagocytosis, etc. The safe concentration on bovine trabecular meshwork cells was 1 × 10-7mg/ml and the LD50 was between 1 × 10-3and 1 × 10-4mg/ml.Conclusions: Refering to previous data, we conclude that conventional clinicalapplication of MMC might do harm to trabecular meshwork cells. Eye Science 2000; 16:38~ 42.

  17. Relationship between lactoferrin, minerals, and somatic cells in bovine milk

    OpenAIRE

    Soyeurt, Hélène; Arnould, Valérie; Bruwier, Damien; Dardenne, Pierre; Romnee, Jean-Michel; Gengler, Nicolas

    2008-01-01

    Selection for increased mastitis resistance is hampered by lack of available data. Currently, somatic cell count or score are proven indicators. However, it should be a priority to increase the number of available indicator traits for mastitis resistance. The aim of this research was to study the relationships among potential indicator traits as lactoferrin content, concentrations of major minerals in milk (calcium, Ca; sodium, Na; phosphore, P), and somatic cell count. Firs...

  18. Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells.

    Science.gov (United States)

    Bai, Chunyu; Li, Xiangchen; Gao, Yuhua; Yuan, Ziao; Hu, Pengfei; Wang, Hui; Liu, Changqing; Guan, Weijun; Ma, Yuehui

    2016-09-01

    Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC-derived pluripotent stem cells (N-iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N-iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR-302/367-mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N-iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis-related genes p53 and p21. Then, N-iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N-iPS (M-N-iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N-iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M-N-iPS via melatonin receptors 1 (MT1). Finally, we verified that N-iPS cells and M-N-iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers. PMID:27090494

  19. Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells.

    Science.gov (United States)

    Bai, Chunyu; Li, Xiangchen; Gao, Yuhua; Yuan, Ziao; Hu, Pengfei; Wang, Hui; Liu, Changqing; Guan, Weijun; Ma, Yuehui

    2016-09-01

    Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC-derived pluripotent stem cells (N-iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N-iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR-302/367-mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N-iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis-related genes p53 and p21. Then, N-iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N-iPS (M-N-iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N-iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M-N-iPS via melatonin receptors 1 (MT1). Finally, we verified that N-iPS cells and M-N-iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.

  20. Zinc supplementation protects against cadmium accumulation and cytotoxicity in Madin-Darby bovine kidney cells.

    Directory of Open Access Journals (Sweden)

    Ding Zhang

    Full Text Available Cadmium ions (Cd2+ have been reported to accumulate in bovine tissues, although Cd2+ cytotoxicity has not been investigated thoroughly in this species. Zinc ions (Zn2+ have been shown to antagonize the toxic effects of heavy metals such as Cd2+ in some systems. The present study investigated Cd2+ cytotoxicity in Madin-Darby bovine kidney (MDBK epithelial cells, and explored whether this was modified by Zn2+. Exposure to Cd2+ led to a dose- and time-dependent increase in apoptotic cell death, with increased intracellular levels of reactive oxygen species and mitochondrial damage. Zn2+ supplementation alleviated Cd2+-induced cytotoxicity and this protective effect was more obvious when cells were exposed to a lower concentration of Cd2+ (10 μM, as compared to 50 μM Cd2+. This indicated that high levels of Cd2+ accumulation might induce irreversible damage in bovine kidney cells. Metallothioneins (MTs are metal-binding proteins that play an essential role in heavy metal ion detoxification. We found that co-exposure to Zn2+ and Cd2+ synergistically enhanced RNA and protein expression of MT-1, MT-2, and the metal-regulatory transcription factor 1 in MDBK cells. Notably, addition of Zn2+ reduced the amounts of cytosolic Cd2+ detected following MDBK exposure to 10 μM Cd2+. These findings revealed a protective role of Zn2+ in counteracting Cd2+ uptake and toxicity in MDBK cells, indicating that this approach may provide a means to protect livestock from excessive Cd2+ accumulation.

  1. Zinc supplementation protects against cadmium accumulation and cytotoxicity in Madin-Darby bovine kidney cells.

    Science.gov (United States)

    Zhang, Ding; Liu, Jingying; Gao, Jianfeng; Shahzad, Muhammad; Han, Zhaoqing; Wang, Zhi; Li, Jiakui; Sjölinder, Hong

    2014-01-01

    Cadmium ions (Cd2+) have been reported to accumulate in bovine tissues, although Cd2+ cytotoxicity has not been investigated thoroughly in this species. Zinc ions (Zn2+) have been shown to antagonize the toxic effects of heavy metals such as Cd2+ in some systems. The present study investigated Cd2+ cytotoxicity in Madin-Darby bovine kidney (MDBK) epithelial cells, and explored whether this was modified by Zn2+. Exposure to Cd2+ led to a dose- and time-dependent increase in apoptotic cell death, with increased intracellular levels of reactive oxygen species and mitochondrial damage. Zn2+ supplementation alleviated Cd2+-induced cytotoxicity and this protective effect was more obvious when cells were exposed to a lower concentration of Cd2+ (10 μM), as compared to 50 μM Cd2+. This indicated that high levels of Cd2+ accumulation might induce irreversible damage in bovine kidney cells. Metallothioneins (MTs) are metal-binding proteins that play an essential role in heavy metal ion detoxification. We found that co-exposure to Zn2+ and Cd2+ synergistically enhanced RNA and protein expression of MT-1, MT-2, and the metal-regulatory transcription factor 1 in MDBK cells. Notably, addition of Zn2+ reduced the amounts of cytosolic Cd2+ detected following MDBK exposure to 10 μM Cd2+. These findings revealed a protective role of Zn2+ in counteracting Cd2+ uptake and toxicity in MDBK cells, indicating that this approach may provide a means to protect livestock from excessive Cd2+ accumulation.

  2. A gene expression programme induced by bovine colostrum whey promotes growth and wound-healing processes in intestinal epithelial cells

    OpenAIRE

    Blais, M.; Pouliot, Y.; Gauthier, S; Boutin, Y.; Lessard, M.

    2014-01-01

    Bovine colostrum is well known for its beneficial properties on health and development. It contains a wide variety of bioactive ingredients that are known to promote a number of cellular processes. Therefore the use of colostrum whey as a feed additive to promote intestinal health has been proposed, yet little is known about mechanisms implicated in its beneficial properties on intestinal epithelial cells. In the present paper, casein were removed from bovine colostrum and the remaining liqui...

  3. Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin

    International Nuclear Information System (INIS)

    The (35S)glycosaminoglycans ((35S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with 35SO4 for 72 h, the (35S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of (35S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of (35S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author)

  4. Comparative study on influence of fetal bovine serum and serum of adult rat on cultivation of newborn rat neural cells

    Directory of Open Access Journals (Sweden)

    Sukach A. N.

    2014-09-01

    Full Text Available Aim. To study the influence of fetal bovine serum and serum of adult rats on behavior of newborn rat isolated neural cells during their cultivation in vitro. Methods. The isolation of neural cells from neonatal rat brain. The determination of the dynamics of cellular monolayer formation. Immunocytochemical staining of cells for β-tubulin III, nestin and vimentin. Results. It has been determined that the addition of serum of adult rats to the cultivation medium creates more favorable conditions for survival, attachment and spread of differentiated, and proliferation of the stem/progenitor neural cells of newborn rats during cultivation in vitro compared with the fetal bovine serum. Conclusions. Using the serum of adult rats is preferable for the cultivation of isolated neural cells of newborn rats compared with the fetal bovine serum.

  5. Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li Qianqian

    2010-10-01

    Full Text Available Abstract Background Spontaneous immortalisation of cultured mammary epithelial cells (MECs is an extremely rare event, and the molecular mechanism behind spontaneous immortalisation of MECs is unclear. Here, we report the establishment of a spontaneously immortalised bovine mammary epithelial cell line (BME65Cs and the changes in gene expression associated with BME65Cs cells. Results BME65Cs cells maintain the general characteristics of normal mammary epithelial cells in morphology, karyotype and immunohistochemistry, and are accompanied by the activation of endogenous bTERT (bovine Telomerase Reverse Transcriptase and stabilisation of the telomere. Currently, BME65Cs cells have been passed for more than 220 generations, and these cells exhibit non-malignant transformation. The expression of multiple genes was investigated in BME65Cs cells, senescent BMECs (bovine MECs cells, early passage BMECs cells and MCF-7 cells (a human breast cancer cell line. In comparison with early passage BMECs cells, the expression of senescence-relevant apoptosis-related gene were significantly changed in BME65Cs cells. P16INK4a was downregulated, p53 was low expressed and Bax/Bcl-2 ratio was reversed. Moreover, a slight upregulation of the oncogene c-Myc, along with an undetectable level of breast tumor-related gene Bag-1 and TRPS-1, was observed in BME65Cs cells while these genes are all highly expressed in MCF-7. In addition, DNMT1 is upregulated in BME65Cs. These results suggest that the inhibition of both senescence and mitochondrial apoptosis signalling pathways contribute to the immortality of BME65Cs cells. The expression of p53 and p16INK4a in BME65Cs was altered in the pattern of down-regulation but not "loss", suggesting that this spontaneous immortalization is possibly initiated by other mechanism rather than gene mutation of p53 or p16INK4a. Conclusions Spontaneously immortalised BME65Cs cells maintain many characteristics of normal BMEC cells and

  6. Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

    Science.gov (United States)

    Roth, D A; Rehemtulla, A; Kaufman, R J; Walsh, C T; Furie, B; Furie, B C

    1993-09-15

    A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase. PMID:8378308

  7. Colloidal Properties of Nanoerythrosomes Derived from Bovine Red Blood Cells.

    Science.gov (United States)

    Kuo, Yuan-Chia; Wu, Hsuan-Chen; Hoang, Dao; Bentley, William E; D'Souza, Warren D; Raghavan, Srinivasa R

    2016-01-12

    Liposomes are nanoscale containers that are typically synthesized from lipids using a high-shear process such as extrusion or sonication. While liposomes are extensively used in drug delivery, they do suffer from certain problems including limited colloidal stability and short circulation times in the body. As an alternative to liposomes, we explore a class of container structures derived from erythrocytes (red blood cells). The procedure involves emptying the inner contents of these cells (specifically hemoglobin) and resuspending the empty structures in buffer, followed by sonication. The resulting structures are termed nanoerythrosomes (NERs), i.e., they are membrane-covered nanoscale containers, much like liposomes. Cryo-transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS) are employed for the first time to study these NERs. The results reveal that the NERs are discrete spheres (∼110 nm diameter) with a unilamellar membrane of thickness ∼4.5 nm. Remarkably, the biconcave disc-like shape of erythrocytes is also exhibited by the NERs under hypertonic conditions. Moreover, unlike typical liposomes, NERs show excellent colloidal stability in both buffer as well as in serum at room temperature, and are also able to withstand freeze-thaw cycling. We have explored the potential for using NERs as colloidal vehicles for targeted delivery. Much like conventional liposomes, NER membranes can be decorated with fluorescent or other markers, solutes can be encapsulated in the cores of the NERs, and NERs can be targeted to specifically bind to mammalian cells. Our study shows that NERs are a promising and versatile class of nanostructures. NERs that are harvested from a patient's own blood and reconfigured for nanomedicine can potentially offer several benefits including biocompatibility, minimization of immune response, and extended circulation time in the body. PMID:26684218

  8. Enzyme separation techniques for the study of growth of cells from layers of bovine dental pulp.

    Science.gov (United States)

    Miller, W A; Everett, M M; Freedman, J T; Feagans, W C; Cramer, J F

    1976-08-01

    Effects of the enzymes trypsin, papain, bromelains and ficin on bovine dental pulp tissue were studied. Minced or whole pulps were subjected to each enzyme at 17 degrees, 20 degrees and 37 degrees C for set time intervals, after which aliquots of supernatant fluid were removed for cell counts and viability tests. Pooled samples were subsequently cultured as monolayers in Eagle's MEM plus 10% calf serum. The dissociation characteristics were quite distinct for each enzyme, although quite similar between minced and whole pulp. A parallel histological study was made of the residual pulp tissue. Ficin was found to be the most suitable enzyme for future studies on the growth of isolated pulp cells from various layers of the bovine pulp, due to its even rate of cell removal, and the good initial viability and subsequent growth of the separated cells in monolayer culture. Further studies on ficin may show that it is more suitable for enzymatic separation of tissues generally than the more commonly used trypsin, a major advantage being its use in media containing Ca2+ and Mg2+.

  9. Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage

    Science.gov (United States)

    Jin, Xiao-Lu; Shen, Xiao-Ge; Sun, Li-Ping; Wu, Li-Ming; Wei, Jiang-Qin; Marcucci, Maria Cristina; Hu, Fu-Liang; Liu, Jian-Xin

    2016-01-01

    Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis. PMID:27433029

  10. FDC-B1: a new monoclonal antibody directed against bovine follicular dendritic cells.

    Science.gov (United States)

    Mélot, F; Defaweux, V; Jolois, O; Collard, A; Robert, B; Heinen, E; Antoine, N

    2004-01-01

    Follicular dendritic cells (FDCs) are a unique population of accessory cells located in the light zone of the germinal centres of lymphoid follicles. Their involvement in the generation of humoral immune responses implies a potential role for these cells in many disorders. Indeed, in prion diseases, FDCs seem to be the major sites of extraneuronal cellular prion protein expression and the principal sites of the infectious agent accumulation in lymphoid organs. The identification of FDC is useful for the analysis of their distribution in reactive lymphoid tissue as well as in pathological conditions. The production and characterisation of a new mouse monoclonal antibody directed against bovine follicular dendritic cells (FDC-B1) is reported. The antigen detected by FDC-B1 is expressed exclusively on the surface of FDCs in ruminant lymphoid organs. The antigen has an approximate molecular weight of 28 kDa. PMID:14700533

  11. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-brain...... barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  12. Genetic assignment of multiple E2 gene products in bovine papillomavirus-transformed cells.

    OpenAIRE

    Lambert, P F; Hubbert, N L; Howley, P M; Schiller, J T

    1989-01-01

    The E2 open reading frame of bovine papillomavirus type 1 has been shown genetically to encode at least three transcriptional regulatory factors, and three E2 specific proteins have been recently identified in virally transformed rodent cells. In this study, the genes encoding these E2 specific proteins have been determined. The 48-kilodalton (kDa) protein was identified as the product of a full-length E2 open reading frame cDNA, which confirmed that this polypeptide is the E2 transactivator....

  13. The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells

    Science.gov (United States)

    KIKU, Yoshio; NAGASAWA, Yuya; TANABE, Fuyuko; SUGAWARA, Kazue; WATANABE, Atsushi; HATA, Eiji; OZAWA, Tomomi; NAKAJIMA, Kei-ichi; ARAI, Toshiro; HAYASHI, Tomohito

    2016-01-01

    Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes. PMID:27211287

  14. Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

    International Nuclear Information System (INIS)

    The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth

  15. Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Rauner, Gat, E-mail: gat.rauner@mail.huji.ac.il [Institute of Animal Science, ARO, The Volcani Center, P.O. Box 6, Bet-Dagan, 50250 (Israel); The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem (Israel); Barash, Itamar, E-mail: itamar.barash@mail.huji.ac.il [Institute of Animal Science, ARO, The Volcani Center, P.O. Box 6, Bet-Dagan, 50250 (Israel)

    2014-10-15

    The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth.

  16. Bases fisiológicas para una interacción entre las células cromafines y las endoteliales de la glándula adrenal Physiological bases for an interaction between chromaffin and endothelial cells from the adrenal gland

    Directory of Open Access Journals (Sweden)

    MARIO LUXORO

    2001-03-01

    Full Text Available En este trabajo tratamos de investigar las posibles interacciones entre las células endoteliales de la glándula adrenal y aquellas sustancias relacionadas con la secreción de las células cromafines. Para lo anterior, estudiamos el efecto de acetilcolina (ACh, o de catecolaminas (CA tanto en el nivel de Ca2+ citoplasmático ([Ca2+]i, como en el potencial de membrana de las células endoteliales. Nuestros resultados muestran que tanto la ACh como la nicotina, pero no la muscarina, son capaces de inducir un aumento del [Ca2+]i y una despolarización de la membrana plasmática de las células endoteliales. El antagonista nicotínico, hexametonium, bloquea tanto el efecto de la ACh como de la nicotina lo que sugiere la presencia de receptores nicotínicos. Por otra parte, las CA (tanto adrenalina como noradrenalina o agonistas a1-adrenérgicos también producen un aumento del [Ca2+]i en las células endoteliales aunque no despolarización evidente. En este caso, el aumento es bifásico siendo la primera fase de un pico rápido e independiente del Ca2+ extracelular en tanto que la segunda se presenta con oscilaciones y depende tanto de que los canales de Ca2+ no estén bloqueados como de la presencia de ese ión en el medio externo. Dado que se ha demostrado que el aumento del [Ca2+]i en las células endoteliales desencadena la secreción de sustancias vasodilatadoras (prostaciclina y óxido nítrico, proponemos que éste sería un mecanismo compensatorio del sistema para contrarestar el enorme efecto vasoconstrictor de las CA secretadas por las células cromafinesIn this work we investigated the possible interactions between the endothelial cells from the adrenal gland and the substances related with the secretion from the chromaffin cells. In order to do so, we studied the effect of acetyl-choline (ACh or of catecholamines (CA on the level of the membrane potential and the cytoplasmic concentration of free calcium ([Ca2+]i in the endothelial

  17. The Effect of Deproteinized Bovine Bone Mineral on Saos-2 Cell Proliferation

    Science.gov (United States)

    Khojasteh, Arash; Ghahremani, Mohammad Hossein; Ostad, Seyed Nasser; Eslami, Mohammad; Motahhary, Pourya; Morad, Golnaz; Shidfar, Shireen

    2013-01-01

    Introduction Deproteinized bovine bone mineral (Bio-Oss) is a xenogenic bone substitute, widely used in maxillofacial bone regeneration. The aim of this in vitro study was to investigate its influence on the growth behavior of human osteosarcoma cell line, Saos-2 culture, and compare it with the physiologic dose of Dexamethasone, an inductive factor for osteoblasts. Materials and Methods Human osteosarcoma cells, Saos-2, were cultured on Bio-Oss and their growth rate was compared to Saos-2 cultures treated with Dexamethasone 10-7 M in contrast to cells cultivated in PBS, in the control group. Assessment of proliferation was performed after 24, 36, and 48 hours by counting cells using trypan blue exclusion method. Alkaline phosphatase was measured spectrophotometrically at 405 nm with paranitrophenol buffer. Results After 48 hours, the number of Saos-2 cells increased significantly when subcultured with Bio-Oss. Bio-Oss was more effective on the enhancement of proliferation of Saos-2 cells when compared to the physiologic dose of Dexamethasone (P<0.05). Alkaline phosphatase activity increased in cells grown on Bio-Oss and dexamethasone 10-7 M in contrast to cells cultivated in PBS control group. The greatest level of activity was observed in the group containing Bio-Oss after 48 hour. Conclusion The significant increase of cell proliferation and alkaline phosphatase activity in cells cultured on Bio-Oss, compared to Dexamethasone-treated cells, suggests the important role of this bone substitute in promoting bone regeneration. PMID:23922573

  18. Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture.

    Science.gov (United States)

    Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

    2016-01-01

    Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium. PMID:27531556

  19. Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin–Darby bovine kidney cell line

    Science.gov (United States)

    Samrath, Devprabha; Shakya, Sanjay; Rawat, Nidhi; Gilhare, Varsha Rani; Singh, Fateh

    2016-01-01

    Aim: Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test. Materials and Methods: Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test. Results: Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test. Conclusion: BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test. PMID:27051213

  20. Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin–Darby bovine kidney cell line

    Directory of Open Access Journals (Sweden)

    Devprabha Samrath

    2016-02-01

    Full Text Available Aim: Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1 from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK cell line. Further, the virus was identified by agar gel immunodiffusion (AGID test. Materials and Methods: Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test. Results: Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test. Conclusion: BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test.

  1. Mitochondrial DNA dynamics during in vitro culture and pluripotency induction of a bovine Rho0 cell line.

    Science.gov (United States)

    Pessôa, L V F; Bressan, F F; Chiaratti, M R; Pires, P R L; Perecin, F; Smith, L C; Meirelles, F V

    2015-10-30

    Large number of cellular changes and diseases are related to mutations in the mitochondrial DNA copy number. Cell culture in the presence of ethidium bromide is a known way of depleting mitochondrial DNA and is a useful model for studying such conditions. Interestingly, the morphology of these depleted cells resembles that of pluripotent cells, as they present larger and fragmented mitochondria with poorly developed cristae. Herein, we aimed to study the mechanisms responsible for the control of mitochondrial DNA replication during mitochondrial DNA depletion mediated by ethidium bromide and during the in vitro induction of cellular pluripotency with exogenous transcription factor expression in a bovine model. This article reports the generation of a bovine Rho0 mesenchymal cell line and describes the analysis of mitochondrial DNA copy number in a time-dependent manner. The expression of apoptosis and mitochondrial-related genes in the cells during mitochondrial DNA repletion were also analyzed. The dynamics of mitochondrial DNA during both the depletion process and in vitro reprogramming are discussed. It was possible to obtain bovine mesenchymal cells almost completely depleted of their mitochondrial DNA content (over 90%). However, the production of induced pluripotent stem cells from the transduction of both control and Rho0 bovine mesenchymal cells with human reprograming factors was not successful.

  2. Cytotoxicity Testing of Temporary Luting Cements with Two- and Three-Dimensional Cultures of Bovine Dental Pulp-Derived Cells

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    Hayriye Esra Ülker

    2013-01-01

    Full Text Available This study evaluated the cytotoxicity of eugenol-containing and eugenol-free temporary luting cements. For cytotoxicity testing, bovine pulp-derived cells transfected with Simian virus 40 Large T antigen were exposed to extracts of eugenol-containing (Rely X Temp E and eugenol-free (Provicol, PreVISION CEM, and Rely X Temp NE temporary luting cements for 24 h. The cytotoxicity of the same materials was also evaluated in a dentin barrier test device using three-dimensional cell cultures of bovine pulp-derived cells. The results of the cytotoxicity studies with two-dimensional cultures of bovine dental pulp-derived cells revealed that cell survival with the extracts of Rely X Temp E, Provicol, PreVISION CEM, and Rely X Temp NE was 89.1%, 84.9%, 92.3%, and 66.8%, respectively. Rely X Temp NE and Provicol showed cytotoxic effects on bovine dental pulp-derived cells (. The results of the dentin barrier test revealed that cell survival with the above-mentioned temporary cement was 101.5%, 91.9%, 93.5%, and 90.6%, respectively. None of the temporary luting cements significantly reduced cell survival compared with the negative control in the dentin barrier test (. Biologically active materials released from temporary luting cements may not influence the dentine-pulp complex if the residual dentine layer is at least 0.5 mm thick.

  3. Isolation and Culture of Bovine Oviductal Epithelial Cells for Use in the Anatomy and Physiology Laboratory and Undergraduate Research

    Science.gov (United States)

    Way, Amy L.

    2006-01-01

    This article presents methods for the isolation and culture of epithelial cells from the bovine oviduct for use in both research and the teaching laboratory and provides examples of ways that an oviductal cell culture can be incorporated into an undergraduate research program. Cow reproductive tracts are readily available from area butchers, and…

  4. Complete Genome Sequence of Noncytopathic Bovine Viral Diarrhea Virus 1 Contaminating a High-Passage RK-13 Cell Line

    OpenAIRE

    Nam, Bora; Li, Ganwu; Zheng, Ying; Zhang, Jianqiang; Shuck, Kathleen M.; Timoney, Peter J.; Balasuriya, Udeni B. R.

    2015-01-01

    A high-passage rabbit kidney RK-13 cell line (HP-RK-13[KY], originally derived from the ATCC CCL-37 cell line) used in certain laboratories worldwide is contaminated with noncytopathic bovine viral diarrhea virus (ncpBVDV). On complete genome sequence analysis, the virus strain was found to belong to BVDV group 1b.

  5. Transcriptional response of HT-29 intestinal epithelial cells to human and bovine milk oligosaccharides.

    Science.gov (United States)

    Lane, Jonathan A; O'Callaghan, John; Carrington, Stephen D; Hickey, Rita M

    2013-12-01

    Human milk oligosaccharides (HMO) have been shown to interact directly with immune cells. However, large quantities of HMO are required for intervention or clinical studies, but these are unavailable in most cases. In this respect, bovine milk is potentially an excellent source of commercially viable analogues of these unique molecules. In the present study, we compared the transcriptional response of colonic epithelial cells (HT-29) to the entire pool of HMO and bovine colostrum oligosaccharides (BCO) to determine whether the oligosaccharides from bovine milk had effects on gene expression that were similar to those of their human counterparts. Gene set enrichment analysis of the transcriptional data revealed that there were a number of similar biological processes that may be influenced by both treatments including a response to stimulus, signalling, locomotion, and multicellular, developmental and immune system processes. For a more detailed insight into the effects of milk oligosaccharides, the effect on the expression of immune system-associated glycogenes was chosen as a case study when performing validation studies. Glycogenes in the current context are genes that are directly or indirectly regulated in the presence of glycans and/or glycoconjugates. RT-PCR analysis revealed that HMO and BCO influenced the expression of cytokines (IL-1β, IL-8, colony-stimulating factor 2 (granulocyte-macrophage) (GM-CSF2), IL-17C and platelet factor 4 (PF4)), chemokines (chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 3 (CXCL3), chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 6 (CXCL6), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X3-C motif) ligand 1 (CX3CL1) and CXCL2) and cell surface receptors (interferon γ receptor 1 (IFNGR1), intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and IL-10 receptor α (IL10RA)). The present study suggests

  6. Intranuclear Localization of EGFP-mouse PPARγ1 in Bovine Fibroblast Cells

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    Sorayya Ghasemi

    2010-01-01

    Full Text Available Objective: The aim of this study was to clone PPARγ1 cDNA in an appropriate mammalianexpression vector, with a chimeric cDNA form, encompassing PPARγ with enhanced greenfluorescent protein (EGFP cDNA. This recombinant plasmid will be used for further analysesto investigate the molecular mechanism of PPARγ1 for neural differentiation process.Moreover, the nuclear localization of the PPARγ1 protein linked to EGFP marker was chasedby using transient transfection of a constructed plasmid into bovine fibroblast cells.Materials and Methods: Total RNA was extracted from the fatty tissue of an adult mouse.Using specific pair primers, PPARγ1 cDNA was synthesized and amplified to producethe entire length of ORF. RT-PCR products containing PPARγ1 cDNA were treated byenzymatic digestion and inserted into the pEGFP-C1 downstream from EGFP cDNA. Theconstructed vector was used for transformation into bacterial competent cells. Positivecolonies which showed inserted PPARγ1 cDNA were selected for plasmid preparationsand additional analysis was performed to ensure that PPARγ1 cDNA was inserted properly.Finally, to confirm the intracellular localization of EGFP-PPARγ1, bovine fibroblastcells were transfected with the recombinant plasmid.Results: Our results from enzymatic digestion and sequencing confirmed, as expected, thatPPARγ1 cDNA was amplified and cloned correctly. This cDNA gene encompassed 1428 bp.The related product was entered into the nucleus of bovine fibroblasts after transfection ofits cDNA.

  7. Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

    Science.gov (United States)

    McGill, Jodi L.; Rusk, Rachel A.; Guerra-Maupome, Mariana; Briggs, Robert E.; Sacco, Randy E.

    2016-01-01

    Human respiratory syncytial virus (HRSV) is a leading cause of severe lower respiratory tract infection in children under five years of age. IL-17 and Th17 responses are increased in children infected with HRSV and have been implicated in both protective and pathogenic roles during infection. Bovine RSV (BRSV) is genetically closely related to HRSV and is a leading cause of severe respiratory infections in young cattle. While BRSV infection in the calf parallels many aspects of human infection with HRSV, IL-17 and Th17 responses have not been studied in the bovine. Here we demonstrate that calves infected with BRSV express significant levels of IL-17, IL-21 and IL-22; and both CD4 T cells and γδ T cells contribute to this response. In addition to causing significant morbidity from uncomplicated infections, BRSV infection also contributes to the development of bovine respiratory disease complex (BRDC), a leading cause of morbidity in both beef and dairy cattle. BRDC is caused by a primary viral infection, followed by secondary bacterial pneumonia by pathogens such as Mannheimia haemolytica. Here, we demonstrate that in vivo infection with M. haemolytica results in increased expression of IL-17, IL-21 and IL-22. We have also developed an in vitro model of BRDC and show that co-infection of PBMC with BRSV followed by M. haemolytica leads to significantly exacerbated IL-17 production, which is primarily mediated by IL-17-producing γδ T cells. Together, our results demonstrate that calves, like humans, mount a robust IL-17 response during RSV infection; and suggest a previously unrecognized role for IL-17 and γδ T cells in the pathogenesis of BRDC. PMID:26942409

  8. Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos.

    Science.gov (United States)

    Arat, Sezen; Caputcu, Arzu Tas; Cevik, Mesut; Akkoc, Tolga; Cetinkaya, Gaye; Bagis, Haydar

    2016-08-01

    This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system. PMID:26444069

  9. Bovine Herpesvirus 1 Protein bICP0 Represses the Transcription of bISG15 in Fetal Bovine Lung Cells

    Institute of Scientific and Technical Information of China (English)

    Chang Liu; Xiao-hong Kong; Wen-tao Qiao; Yun-qi Geng

    2011-01-01

    The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells.Bovine Herpesvirus 1(BHV-1),which is a viral pathogen of cattle,can infect FBL cells and induce cytopathic effects.Real-time PCR assays showed that BHV- 1 's infection could repress the basal or inducible transcription of bISG15 in FBL cells.It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis.Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG 15 in FBL cells,so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed.The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3.Taken together,our work suggested that BHV-I had some molecular mechanism to resist the cellular bISG15'santiviral functions.

  10. PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

    Science.gov (United States)

    Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

    2016-08-01

    Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle. PMID:27260975

  11. Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

    KAUST Repository

    Janjanam, Jagadeesh

    2013-10-01

    Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Increase in proto-oncogene mRNA transcript levels in bovine lymphoid cells infected with a cytopathic type 2 bovine viral diarrhea virus.

    Science.gov (United States)

    Neill, John D; Ridpath, Julia F

    2008-08-01

    Infection of susceptible animals with bovine viral diarrhea viruses (BVDV) can result in an array of disease symptoms that are dependent in part on the strain of infecting virus and the physiological status of the host. BVDV are lymphotrophic and exist as two biotypes. Cytopathic BVDV kill cells outright while noncytopathic strains can readily establish persistent infections. The molecular mechanisms behind these different affects are unknown. To gain a better understanding of the mechanisms of disease, serial analysis of gene expression (SAGE), a powerful method for global gene expression analysis, was employed to examine gene expression changes in BVDV-infected BL3 cells, a bovine B-cell lymphosarcoma cell line. SAGE libraries were constructed from mRNA derived from BL3 cells that were noninfected or infected with the cytopathic BVDV2 strain 296c. Annotation of the SAGE data showed the expression of many genes that are characteristic of B cells and integral to their function. Comparison of the SAGE databases also revealed a number of genes that were differentially expressed. Of particular interest was the increased numbers of transcripts encoding proto-oncogenes (c-fos, c-jun, junB, junD) in 296c-infected cells, all of which are constituents of the AP-1 transcriptional activation complex. Real-time RT-PCR confirmed these results and indicated that the actual increases were larger than that predicted by SAGE. In contrast, there was no corresponding increase in protein levels, but instead a significant decrease of c-jun and junB protein levels in the infected BL3 cells was observed. Rather than an increase in transcription of these genes, it appeared that these proto-oncogenes transcripts accumulated in the BVDV2-infected cells.

  13. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    International Nuclear Information System (INIS)

    Incubation of [35S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [35S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [35S]heparin proteoglycan after 24 hours and the appearance of free [35S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [35S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule

  14. Effect of anabolics on bovine granulosa-luteal cell primary cultures.

    Directory of Open Access Journals (Sweden)

    Bartolomeo Biolatti

    2007-10-01

    Full Text Available Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione. Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.

  15. Dynamics of mercury, cadmium and vanadium in cultured bovine kidney cells: an examination of relationships to cytotoxicity and cell function

    International Nuclear Information System (INIS)

    The objective of this study was to partially define the in vitro cellular response to mercury, cadmium and vanadium insult. A bovine kidney cell line served as the model system for examining the relationship of the cellular dynamics of metal accumulation and distribution to cytotoxicity. Additionally, biochemical marker functions were monitored in surviving cells to determine the importance of metal uptake and distribution to cell functionality. Each metal (HgCl2, CdCl2, and Na3VO4) elicited a concentration-related cytotoxicity which was correlated to the cellular metal burden. Multiphasic accumulation kinetics were established for mercury and vanadium; cadmium was accumulated in a linear fashion. Subcellular metal distribution was independent of both the extra-cellular metal concentration and the degree of cytotoxicity. Biochemical marker functions indicated a toxicity-related decrease in cell functionality in surviving cells for all metals

  16. Effect of corticosteroid binding proteins on the steroidogenic activity of bovine adrenocortical cell suspensions.

    Science.gov (United States)

    Basset, M; Rostaing-Metz, B; Chambaz, E M

    1982-07-01

    The possible role of steroid binding proteins in the hormonal secretion process of a steroidogenic tissue was examined using bovine adrenocortical cell suspensions, either under basal conditions or in the presence of half-maximally active concentration (1 x 10(-9) M) of synthetic adrenocorticotropic hormone (ACTH). Three types of plasma cortisol binding proteins were used, namely bovine serum albumine (BSA), purified transcortin (CBG) and purified anticortisol immunoglobulins (IgG). When added to the incubation medium, CBG (at 1 x 10(-10) to 2 x 10(-9) M cortisol binding sites) and anticortisol IgG (at 4.8 x 10(-10) to 3 x 10(-9) M cortisol binding sites) did not influence either the basal nor the ACTH-stimulated net cortisol production of the cell preparations. Whereas crystallized and delipidated BSA showed also no effect, crude commercial BSA preparation (Cohn fraction V) exhibited an ACTH-like cofactor effect which resulted in a marked increase in the net cortisol production by stimulated cells. These observations might be explained by the presence in crude BSA of lipoprotein-cholesterol complexes, possibly acting as an extracellular source of cholesterol available for corticosteroidogenesis. It may be concluded that specific high affinity cortisol binding systems present outside adrenocortical steroidogenic cells do not influence their secretory activity under short term in vitro condition. In addition, it can be stressed that use of ill defined protein preparations (e.g. crude BSA) may lead to artifactual observations in the study of the differentiated functions of isolated steroidogenic cells. PMID:6287106

  17. Bovine herpesvirus type 4 infection modulates autophagy in a permissive cell line.

    Science.gov (United States)

    Montagnaro, Serena; Ciarcia, Roberto; Pagnini, Francesco; De Martino, Luisa; Puzio, Maria Valeria; Granato, Giovanna Elvira; Avino, Franca; Pagnini, Ugo; Iovane, Giuseppe; Giordano, Antonio

    2013-07-01

    Bovine herpesvirus type 4 (BoHV-4), like other herpesviruses, induces a series of alterations in the host cell that modify the intracellular environment in favor of viral replication, survival and spread. This research examined the impact of BoHV-4 infection on autophagy in BoHV-4 infected Madin Darby bovine kidney (MDBK) cells. Protein extracts of BoHV-4 infected and control MDBK cells were subjected to Western blot. The concentrations of the autophagy and apoptosis-related proteins Beclin 1, p21, PI3 kinase, Akt1/2, mTOR, phospho mTOR, p62 and the light chain three (LC3) were normalized to the actin level and expressed as the densitometric ratio. Western blot analysis of virus-infected cells revealed that autophagic degradation pathway was induced in the late phase of BoHV-4 infection. After 48 h post-infection the protein LC3II, which is essential for autophagy was found to be markedly increased, while infection of MDBK cells with BoHV-4 resulted in a depletion of p62 levels. Becline 1, PI3 kinase, Akt1/2 and p21 expression increased between 24 and 48 h post-infection. Surprisingly, mTOR and its phosphorylated form, which are negative regulators of autophagy, also increased after 24 h post-infection. In conclusion, our findings suggest that BoHV-4 has developed mechanisms for modulation of autophagy that are probably part of a strategy designed to enhance viral replication and to evade the immune system. Additional studies on the relationship between autophagy and BoHV-4 replication and survival, in both lytic and latent replication phases, are needed to understand the role of autophagy in BoHV-4 pathogenesis.

  18. Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

    Directory of Open Access Journals (Sweden)

    Griffiths T Daniel

    2009-05-01

    Full Text Available Abstract Background The Retinal Pigmented Epithelium (RPE is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD, Retinitis Pigmentosa (RP, Fundus Flavimaculatus (FFM, Best's disease and Age-related Macular Degeneration (AMD. We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases. Results The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency. Conclusion The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal

  19. Phenotypic characterization of bovine memory cells responding to mycobacteria in IFNγ enzyme linked immunospot assays.

    Science.gov (United States)

    Blunt, Laura; Hogarth, Philip J; Kaveh, Daryan A; Webb, Paul; Villarreal-Ramos, Bernardo; Vordermeier, Hans Martin

    2015-12-16

    Bovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells. Using expression of CD45RO and CD62L we purified by cytometric cell sorting four distinct CD4(+) populations: CD45RO(+)CD62L(hi), CD45RO(+)CD62L(lo), CD45RO(-)CD62L(hi) and CD45RO(-)CD62L(lo) (although due to low and inconsistent cell recovery, this population was not considered further in this study), in BCG vaccinated and Mycobacterium bovis infected cattle. These populations were then tested in the cELISPOT assay. The main populations contributing to production of IFNγ in the cELISPOT were of the CD45RO(+)CD62L(hi) and CD45RO(+)CD62L(lo) phenotypes. These cell populations have been described in other species as central and effector memory cells, respectively. Following in vitro culture and flow cytometry we observed plasticity within the bovine CD4(+) T-cell phenotype. Populations switched phenotype, increasing or decreasing expression of CD45RO and CD62L within 24h of in vitro stimulation. After 14 days all IFNγ producing CD4(+) T cells expressed CD45RO regardless of the original phenotype of the sorted population. No differences were detected in behavior of cells derived from BCG-vaccinated animals compared to cells derived from naturally infected animals. In conclusion, although multiple populations of CD4(+) T memory cells from both BCG vaccinated and M. bovis infected animals contributed to cELISPOT responses, the dominant contributing population consists of central-memory-like T cells (CD45RO(+)CD62L(hi)).

  20. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  1. Interactions of bovine viral diarrhoea virus glycoprotein E(rns) with cell surface glycosaminoglycans.

    Science.gov (United States)

    Iqbal, M; Flick-Smith, H; McCauley, J W

    2000-02-01

    Recombinant E(rns) glycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble E(rns) to the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged E(rns) to permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. E(rns) failed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. E(rns) also bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of E(rns) binding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-L-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of E(rns) to cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction. PMID:10644844

  2. Effects of donor cells on in vitro development of cloned bovine embryos

    Institute of Scientific and Technical Information of China (English)

    Jing Fu; Pengfei Guan; Leiwen Zhao; Hua Li; Shuzhen Huang; Fanyi Zeng; Yitao Zeng

    2008-01-01

    The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The ooeytes were obtained through ovum pick up operation. After in vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer.The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastoeyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129)had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P0.05). It was concluded that the genetic background of the donor cells could affect the effi-ciency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.

  3. Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization, and multipotentiality.

    Science.gov (United States)

    Sampaio, R V; Chiaratti, M R; Santos, D C N; Bressan, F F; Sangalli, J R; Sá, A L A; Silva, T V G; Costa, N N; Cordeiro, M S; Santos, S S D; Ambrosio, C E; Adona, P R; Meirelles, F V; Miranda, M S; Ohashi, O M

    2015-01-15

    Adult stem cells are known for their plasticity and their potential to differentiate into several different cell types; these characteristics have implications for cell therapy and reproductive biotechnologies. In this study, we report on the isolation and characterization of mesenchymal stem cells (MSC) derived from bovine and buffalo adipose tissue. Cells isolated using enzymatic digestion of bovine and buffalo adipose-tissue biopsy samples were grown in vitro for at least 15 passages, verifying their capacity to proliferate. These cells were also subjected to immunophenotypic characterization for the presence of CD90, CD105, and CD79, and the absence of CD45, CD34, and CD73, which are positive and negative markers of MSC, respectively. To prove their multipotency, the cells were induced to differentiate into three different cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (Chondrogenic-Alcian Blue, Osteogenic-Alizarin Red, and Adipogenic-Oil-Red O, respectively) to confirm differentiation. Gene expression analysis of pluripotency-related genes was also conducted. Our results suggest that adipose tissue from bovines and buffalos can be used as a source of MSC, making adipose tissue-derived cells an interesting option for cell therapy and regenerative medicine. Additionally, these findings have implications for reproductive biotechnology because the use of MSC as nuclear donors has been linked to an increase in the efficiency of nuclear transfer.

  4. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  5. Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Shaokun Zhang

    2016-01-01

    Full Text Available The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration.

  6. 奶牛乳腺上皮细胞的原代培养%Primary Culture of Bovine Mammary Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    吴娟; 王凤龙; 王申元

    2009-01-01

    [Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2-4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

  7. Biochemical responsiveness of a bovine kidney cell line to inorganic mercury

    Energy Technology Data Exchange (ETDEWEB)

    Bracken, W.M.; Sharma, R.P.

    1985-07-01

    Mercury compounds are known environmental pollutants. A bovine kidney cell line (MDBK) was used to assess the importance of a variety of biochemical cell functions to mercuric chloride (Hg) cytotoxicity. Mercury concentrations utilized ranged between 1-10 ..mu..M and elicited 0-45% cytotoxicity at 24-hr post-exposure. Leucine incorporation into cellular protein decreased in a concentration-dependent manner which paralleled the cytotoxicity. Thymidine incorporation into deoxyribonucleic acid was less sensitive to Hg inhibition. K..mu..-dependent phosphatase (KP), succinate dehydrogenase (SDH) and acid phosphatase (AP) were monitored in surviving cells 24 hr post-exposure and in a cell-free system. The activity of KP was reduced to 50% of control activity following 24 hr exposure of MDBK cells to Hg. The activities of SDH and AP of treated cultures remained at control levels. Measurement of reduced glutathione after 24-hr treatment indicated a marginal elevation (120-176%). In a cell-free system, KP, SDH and AP were sensitive to Hg to varying degrees; KP and SDH were most sensitive with IC50s of 1 and 10 ..mu..M Hg, respectively. The importance of biochemical changes in relation to the developing cytotoxicity are discussed.

  8. Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.

    Science.gov (United States)

    Park, Min Jee; Lee, Seung Eun; Kim, Eun Young; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2015-06-01

    Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (pEVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques. PMID:25984830

  9. Bovine Muc1 inhibits binding of enteric bacteria to Caco-2 cells.

    Science.gov (United States)

    Parker, Phillip; Sando, Lillian; Pearson, Roger; Kongsuwan, Kritaya; Tellam, Ross L; Smith, Stuart

    2010-01-01

    Inhibition of bacterial adhesion to intestinal epithelial receptors by the consumption of natural food components is an attractive strategy for the prevention of microbial related gastrointestinal illness. We hypothesised that Muc1, a highly glycosylated mucin present in cows' milk, may be one such food component. Purified bovine Muc1 was tested for its ability to inhibit binding of common enteric bacterial pathogens to Caco-2 cells grown in vitro. Muc1 caused dose-dependent binding inhibition of Escherichia coli, Salmonella enterica serovar Typhimurium (S. Typhimurium), Staphylococcus aureus and Bacillus subtilis. This inhibition was more pronounced for the Gram negative compared with Gram positive bacteria. It was also demonstrated that Muc1, immobilised on a membrane, bound all these bacterial species in a dose-dependent manner, although there was greater interaction with the Gram negative bacteria. A range of monosaccharides, representative of the Muc1 oligosaccharide composition, were tested for their ability to prevent binding of E. coli and S. Typhimurium to Caco-2 cells. Inhibition was structure dependent with sialic acid, L(-) fucose and D(+) mannose significantly inhibiting binding of both Gram negative species. N-acetylglucosamine and N-acetylgalactosamine significantly inhibited binding of E. coli whilst galactose, one of the most abundant Muc1 monosaccharides, showed the strongest inhibition against S. Typhimurium. Treatment with sialidase significantly decreased the inhibitory properties of Muc1, demonstrating the importance of sialic acid in adhesion inhibition. It is concluded that bovine Muc1 prevents binding of bacteria to human intestinal cells and may have a role in preventing the binding of common enteropathogenic bacteria to human intestinal epithelial surfaces.

  10. The Experimental Study of Constructing Tissue Engineered Bone by Compounding Zinc-sintered Bovine Cancellous Bone with Marrow Stromal Cells

    Institute of Scientific and Technical Information of China (English)

    ZHENG Qi-xin; HAO Jie; GUO Xiao-dong; LIU Su-nan; Wu Yong-chao; YAN Yu-hua

    2004-01-01

    To study the osteogenic ability of tissue-engineered bone constructed by compounding zinc-sin-tered bovine cancellous bone with rabbit marrow stromal cells ( MSCs ) in vivo, the zinc- sintered bovine cancellousbone of beta-tricalcium phosphate (TCP) type was prepared by sintering the fresh calf cancellous bone twice andthen loading it with zinc-ion. The rabbit MSCs were cultured, induced and seeded onto the zinc- sintered bovine can-cellous bones. The tissue-engineered bones were then implanted into the rabbits' bock muscles. The newly formedbone tissues were observed by histological methods and the areas of new osseous tissues were measured at the end ofthe 4 th and 8 th week. The zinc-sintered bovine cancellous bones alone were implanted on the other side as control.The osteogenic activity of MSCs was identified by alkaline phosphatase (ALP) staining and calcification nod chi-nalizarin staining. At the end of 4th week, a small amount of new bone tissues was observed. At the end of 8thweek, there were many newly formed bone mature tissues. Moreover, the area of the latter was significantly largerthan that of the former( P<0.01), while in the control group there was no new bone formation. The tissue-engi-neered bone, which was constructed by combining zinc-sintered bovine cancellous bone with MSCs, has satisfactoryosteogenic capabilities in vivo.

  11. Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

    Science.gov (United States)

    Nel-Themaat, L.; Gomez, M.C.; Pope, C.E.; Lopez, M.; Wirtu, G.; Jenkins, J.A.; Cole, A.; Dresser, B.L.; Bondioli, K.R.; Godke, R.A.

    2008-01-01

    The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had ???8 cells at 84 hpa, while 32% of the bovine NT embryos had ???8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had ???8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further. ?? 2008 Mary Ann Liebert, Inc.

  12. Bovine and soybean milk bioactive compounds: Effects on inflammatory response of human intestinal Caco-2 cells.

    Science.gov (United States)

    Calvello, Rosa; Aresta, Antonella; Trapani, Adriana; Zambonin, Carlo; Cianciulli, Antonia; Salvatore, Rosaria; Clodoveo, Maria Lisa; Corbo, Filomena; Franchini, Carlo; Panaro, Maria Antonietta

    2016-11-01

    In this study the effects of commercial bovine and soybean milks and their bioactive compounds, namely genistein, daidzein and equol, on the inflammatory responses induced by lipopolysaccharide (LPS) treatment of human intestinal Caco-2 cells were examined, in terms of nitric oxide (NO) release and inducible nitric oxide synthetase (iNOS) expression. Both milks and their bioactive compounds significantly inhibited, dose-dependently, the expression of iNOS mRNA and protein, resulting in a decreased NO production. The NF-κB activation in LPS-stimulated intestinal cells was also examined. In all cases we observed that cell pre-treatment before LPS activation inhibited the IkB phosphorylation. Accordingly, quantification of bioactive compounds by solid phase microextraction coupled with liquid chromatography has shown that they were absorbed, metabolized and released by Caco-2 cells in culture media. In conclusion, we demonstrated that milks and compounds tested are able to reduce LPS-induced inflammatory responses from intestinal cells, interfering with NF-kB dependent molecular mechanisms. PMID:27211648

  13. In Vitro Developmental Potential of Cloned Embryos Derived from Bovine Somatic Cells and Rabbits Oocyte

    Institute of Scientific and Technical Information of China (English)

    LIU Ya; LI Bin; ZHAO Huan; CHENG Li-zi; ZHANG Xiao-rong; CHEN Da-yuan; ZHANG Yun-hai; ZHANG Zhi-guo; JING Ren-tao; WANG Cun-li; ZHANG Mei-lin; LI Dong-wei

    2003-01-01

    180 reconstituted embryos were produced by nuclear transplantation using bovine ear fibroblasts at G0 or non-G0 stage as donor nuclei and oocytes collected from superovulated multiparous or young rabbits as recipients. After cultivation in two kinds of medium M199+ 10%FBS or RD+ 10%FBS, 112 of them developed to 2-cell stage (62.2%) and 26 to morula stage (14.4%) and 20 of them eventually developed to blastocyst stage (11. 1% ). There is no significant difference for the cleavage rates in two groups of reconstituted embryos derived from G0-stage and non-G0 stage donor cells respectively. However, G0-stage donor cells could result in higher rate of 8-cell - 16-cell stage embryos significantly (P<0.05), as well as higher rate of blastocysts (P<0.01). It seems that using two different culture systems had no significant effects on the cleavage rate, morula rate or blastocyst rate (P>0.05).

  14. Morphological and biological characterization of cell line developed from bovine Echinococcus granulosus.

    Science.gov (United States)

    Echeverría, Claudia I; Isolabella, Dora M; Prieto Gonzalez, Elio A; Leonardelli, Araceli; Prada, Laura; Perrone, Alina; Fuchs, Alicia G

    2010-10-01

    The taeniid tapeworm Echinococcus granulosus is the causative agent of echinococcal disease, a major zoonosis with worldwide distribution. Several efforts to establish an in vitro model of E. granulosus have been undertaken; however, many of them have been designed for Echinococcus multilocularis. In the present study, we have described and characterized a stable cell line obtained from E. granulosus bovine protoscoleces maintained 3 yr in vitro. Growth characterization, morphology by light, fluorescent and electronic microscopy, and karyotyping were carried out. Cell culture origin was confirmed by immunofluorescent detection of AgB4 antigen and by PCR for the mitochondrial cytochrome c-oxidase subunit 1 (DCO1) gene. Cells seeded in agarose biphasic culture resembled a cystic structure, similar to the one formed in secondary hosts. This cell line could be a useful tool to research equinococcal behavior, allowing additional physiological and pharmacological studies, such as the effect of growth factors, nutrients, and antiparasitic drugs on cell viability and growth and on cyst formation.

  15. Cellular distribution of inorganic mercury and its relation to cytotoxicity in bovine kidney cell cultures

    International Nuclear Information System (INIS)

    A bovine kidney cell culture system was used to assess what relationship mercuric chloride (HgCl2) uptake and subcellular distribution had to cytotoxicity. Twenty-four-hour incubations with 0.05-50 μM HgCl2 elicited a concentration-related cytotoxicity. Cellular accumulation of 203Hg was also concentration-related, with 1.0 nmol/106 cells at the IC50. Measurement of Hg uptake over the 24-h exposure period revealed a multiphasic process. Peak accumulation was attained by 1 h and was followed by extrusion and plateauing of intracellular Hg levels. Least-squares regression analysis of the cytotoxicity and cellular uptake data indicated a potential relationship between the Hg uptake and cytotoxicity. However, the subcellular distribution of Hg was not concentration-related. Mitochondria and soluble protein fractions accounted for greater than 65% of the cell-associated Hg at all concentrations. The remaining Hg was distributed between the microsomal (6-10%) and nuclear and cell debris (11-22%) fractions at all concentrations tested. Less than 20% of the total cell-associated Hg was bound with metallothionein-like protein. 31 references, 4 figures, 3 tables

  16. Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.

    Science.gov (United States)

    Wei, Zhiyun; Batagov, Arsen O; Carter, David R F; Krichevsky, Anna M

    2016-01-01

    Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA. PMID:27503761

  17. Removal of glycosaminoglycans from bovine granulosa cells contributes to increased binding of hydrogen-3 heparin

    Energy Technology Data Exchange (ETDEWEB)

    Ax, R.L.; Stodd, C.M.; Boehm, S.K.; Bellin, M.E.

    1986-02-01

    Granulosa cells from small or large bovine follicles were pretreated with enzymes that hydrolyze various glycosaminoglycans, and binding of (/sup 3/H)-heparin to the granulosa was measured. Binding of (/sup 3/H) heparin increased significantly after enzymatic pretreatments with chondroitinase ABC and fungal hyaluronidase, and similar results were obtained with granulosa from small and large follicles. No changes in binding of (/sup 3/H) heparin were detected after hydrolyses with chondroitinase AC and heparinase in either follicle size. Heparitinase, which hydrolyzes heparan sulfate, led to a significant 50% increase in binding of (/sup 3/H) heparin to granulosa from large follicles but was without effect in small follicles. These results suggest that the lower binding of (/sup 3/H) heparin, which has been reported with follicular enlargement, may be due to heparan sulfate occupying or obstructing binding sites for heparin on granulosa from large follicles.

  18. Chitosan modification of adenovirus to modify transfection efficiency in bovine corneal epithelial cells.

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    I-Jong Wang

    Full Text Available BACKGROUND: The purpose of this study is to modulate the transfection efficiency of adenovirus (Ad on the cornea by the covalent attachment of chitosan on adenoviral capsids via a thioether linkage between chitosan modified with 2-iminothiolane and Ad cross-linked with N-[gamma-maleimidobutyryloxy]succinimide ester (GMBS. METHODOLOGY/PRINCIPAL FINDINGS: Modified Ad was obtained by reaction with the heterobifunctional crosslinking reagent, GMBS, producing maleimide-modified Ad (Ad-GMBS. Then, the chitosan-SH was conjugated to Ad-GMBS via a thioether bond at different ratios of Ad to GMBS to chitosan-SH. The sizes and zeta potentials of unmodified Ad and chitosan-modified Ads were measured, and the morphologies of the virus particles were observed under transmission electron microscope. Primary cultures of bovine corneal epithelial cells were transfected with Ads and chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Chitosan modification did not significantly change the particle size of Ad, but the surface charge of Ad increased significantly from -24.3 mV to nearly neutral. Furthermore, primary cultures of bovine corneal epithelial cells were transfected with Ad or chitosan-modified Ad in the absence or presence of anti-Ad antibodies. The transfection efficiency was attenuated gradually with increasing amounts of GMBS. However, incorporation of chitosan partly restored transfection activity and rendered the modified antibody resistant to antibody neutralization. CONCLUSIONS/SIGNIFICANCE: Chitosan can provide a platform for chemical modification of Ad, which offers potential for further in vivo applications.

  19. Novel CD8(+) cytotoxic T cell epitopes in bovine leukemia virus with cattle.

    Science.gov (United States)

    Bai, Lanlan; Takeshima, Shin-Nosuke; Isogai, Emiko; Kohara, Junko; Aida, Yoko

    2015-12-16

    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine. PMID:26552001

  20. Novel CD8(+) cytotoxic T cell epitopes in bovine leukemia virus with cattle.

    Science.gov (United States)

    Bai, Lanlan; Takeshima, Shin-Nosuke; Isogai, Emiko; Kohara, Junko; Aida, Yoko

    2015-12-16

    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine.

  1. Inhibitory effect of extracts of Ginkgo biloba leaves on VEGF-induced hyperpermeability of bovine coronary endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yan QIU; Yao-cheng RUI; Tie-jun LI; Li ZHANG; Peng-yuan YANG

    2004-01-01

    AIM: To study whether extract of Ginkgo biloba (EGb) can protect against atherosclerosis. METHODS: Confluent monolayers of bovine coronary endothelial cells (BCECs), bovine coronary smooth muscle cells (BCSMCs), and cocultures of the two were incubated with medium containing VEGF and/or EGb, and flux of 125Ⅰ-labeled oxidized low density lipoprotein (ox-LDL) across the monolayers was measured. RESULTS: Incubation with VEGF significantly increased the permeability of BCEC monolayers to 125Ⅰ-ox-LDL in a time- and concentration-dependent manner, but had no effect on permeability of BCSMCs or endothelial cells-smooth muscle cells cocultures. EGb significantly inhibited the VEGF-induced hyperpermeability of BCECs. CONCLUSION: VEGF was important in the formation and development of atherosclerosis. The inhibition of VEGF-induced permeability by EGb suggests that extracts of Ginkgo biloba leaves may have important clinical applications in the treatment of cardiovascular diseases.

  2. Tudor-SN Regulates Milk Synthesis and Proliferation of Bovine Mammary Epithelial Cells

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    Jinxia Ao

    2015-12-01

    Full Text Available Tudor staphylococcal nuclease (Tudor-SN is a highly conserved and ubiquitously expressed multifunctional protein, related to multiple and diverse cell type- and species-specific cellular processes. Studies have shown that Tudor-SN is mainly expressed in secretory cells, however knowledge of its role is limited. In our previous work, we found that the protein level of Tudor-SN was upregulated in the nucleus of bovine mammary epithelial cells (BMEC. In this study, we assessed the role of Tudor-SN in milk synthesis and cell proliferation of BMEC. We exploited gene overexpression and silencing methods, and found that Tudor-SN positively regulates milk synthesis and proliferation via Stat5a activation. Both amino acids (methionine and estrogen triggered NFκB1 to bind to the gene promoters of Tudor-SN and Stat5a, and this enhanced the protein level and nuclear localization of Tudor-SN and p-Stat5a. Taken together, these results suggest the key role of Tudor-SN in the transcriptional regulation of milk synthesis and proliferation of BMEC under the stimulation of amino acids and hormones.

  3. Biphasic effect of aspirin on apoptosis of bovine vascular endothelial cells and its molecular mechanism

    Institute of Scientific and Technical Information of China (English)

    Qing-quan CHEN; Wen-lan LIU; Xun GUO; Yuan-jian LI; Zhao-gui GUO

    2007-01-01

    Aim: To investigate the effect of aspirin on the apoptosis of cultured bovine aortic endothelial cells (BAEC) and the signal pathways involved in this process.Methods: BAEC were cultured and passaged in Dulbecco's modified Eagle's medium culture medium. Morphologic changes and quantification of apoptotic cells were determined using fluorescence microscope after staining the cells with Hoechst 33258. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. DNA fragmentation was visualized by agarose gel electrophoresis. Phospho-p38 mitogen-activated protein kinase(MAPK) expression was detected by Western blotting. Results: Aspirin at low concentrations from 1×10-10 mol/L to 1×10-8 mol/L decreased the apoptosis and p38 MAPK phosphorylation induced by H2O2 in BAEC, while high doses of aspi-fin (1×10-7-1×10-4 mol/L) induced typical apoptotic changes in BAEC and stimu-lated the expression of phospho-p38 MAPK in a concentration-dependent manner.SB203580, a specific p38 MAPK inhibitor, blocked such effects. Conclusion:Aspirin exhibits a biphasic effect on the apoptosis in BAEC, reducing apoptosis at low concentration and inducing apoptosis at high concentration, p38 MAPK may be an important signal molecule mediating the effects of aspirin.

  4. Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells

    Science.gov (United States)

    Fujie, Tomoya; Murakami, Masaki; Yoshida, Eiko; Yasuike, Shuji; Kimura, Tomoki; Fujiwara, Yasuyuki; Yamamoto, Chika; Kaji, Toshiyuki

    2016-01-01

    Vascular endothelial cells cover the luminal surface of blood vessels and contribute to the prevention of vascular disorders such as atherosclerosis. Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, inducible protein, which protects cells from the toxicity of heavy metals and active oxygen species. Endothelial MT is not induced by inorganic zinc. Adequate tools are required to investigate the mechanisms underlying endothelial MT induction. In the present study, we found that an organoantimony compound, tris(pentafluorophenyl)stibane, induces gene expression of MT-1A and MT-2A, which are subisoforms of MT in bovine aortic endothelial cells. The data reveal that MT-1A is induced by activation of both the MTF-1–MRE and Nrf2–ARE pathways, whereas MT-2A expression requires only activation of the MTF-1–MRE pathway. The present data suggest that the original role of MT-1 is to protect cells from heavy metal toxicity and oxidative stress in the biological defense system, while that of MT-2 is to regulate intracellular zinc metabolism. PMID:27563876

  5. Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats.

    Science.gov (United States)

    Kim, Sung Eun; Ko, Il Gyu; Shin, Mal Soon; Kim, Chang Ju; Ko, Young Gwan; Cho, Hanjin

    2012-08-01

    Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism. Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyl-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death. PMID:25624793

  6. Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats☆

    Science.gov (United States)

    Kim, Sung Eun; Ko, Il Gyu; Shin, Mal Soon; Kim, Chang Ju; Ko, Young Gwan; Cho, Hanjin

    2012-01-01

    Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism. Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyl-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death. PMID:25624793

  7. Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Stepp, M.A.

    1986-01-01

    Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of (/sup 3/H)-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the ..cap alpha..1 and ..cap alpha..2 chains of type I and the ..cap alpha..1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells.

  8. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

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    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  9. Expressed sequence tags for bovine muscle satellite cells, myotube formed-cells and adipocyte-like cells.

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    Eun Ju Lee

    Full Text Available BACKGROUND: Muscle satellite cells (MSCs represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs and adipocyte-like cells (ALCs, we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs. RESULTS: A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248 highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2 during myogenesis and hemoglobin subunit alpha2 (HBA2 during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay. CONCLUSION: In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to

  10. Three-dimensional model of angiogenesis: coculture of human retinal cells with bovine aortic endothelial cells in the NASA bioreactor.

    Science.gov (United States)

    Dutt, Kamla; Sanford, Gary; Harris-Hooker, Sandra; Brako, Lawrence; Kumar, Ravindra; Sroufe, Angela; Melhado, Caroline

    2003-10-01

    Ocular angiogenesis is the leading cause of blindness and is associated with diabetic retinopathy and age-related macular degeneration. We describe, in this report, our preliminary studies using a horizontally rotating bioreactor (HRB), developed by the National Aeronautics and Space Administration (NASA), to explore growth and differentiation-associated events in the early phase of ocular angiogenesis. Human retinal (HRet) cells and bovine endothelial cells (ECs) were cocultured on laminin-coated Cytodex-3 microcarrier beads in an HRB for 1-36 days. Endothelial cells grown alone in the HRB remained cuboidal and were well differentiated. However, when HRet cells were cocultured with ECs, cordlike structures formed as early as 18-36 h and were positive for von Willebrand factor. In addition to the formation of cords and capillary-like structures, ECs showed the beginning of sprouts. The HRB seems not only to promote accelerated capillary formation, but also to enhance differentiation of retinal precursor cells. This leads to the formation of rosette-like structures (which may be aggregates of photoreceptors that were positive for rhodopsin). Upregulation of vascular endothelial growth factor and basic fibroblast growth factor was seen in retinal cells grown in the HRB as compared with monolayers and could be one of the factors responsible for accelerated capillary formation. Hence, the HRB promotes three-dimensional assembly and differentiation, possibly through promoting cell-to-cell interaction and/or secretion of growth and differentiation factors. PMID:14633374

  11. Short communication: opposing effects of lactoferrin on the proliferation of fibroblasts and epithelial cells from bovine mammary gland.

    Science.gov (United States)

    Nakajima, K; Itoh, F; Nakamura, M; Kawamura, A; Yamazaki, T; Kozakai, T; Takusari, N; Ishisaki, A

    2015-02-01

    Lactoferrin is present in several physiologic fluids, including milk and colostrum. Recently, evidence has accumulated that lactoferrin acts as a regulator of cell proliferation. Lactoferrin mRNA and protein levels in bovine mammary glands are known to markedly increase after cessation of milking. To clarify the role of bovine lactoferrin (bLF) in mammary involution and remodeling during dry periods, we investigated whether bLF affects the proliferation of cultured cells derived from bovine mammary gland and examined the mechanism underlying the proliferative response to bLF. Addition of bLF to the culture medium increased the proliferation of bovine mammary stromal fibroblasts (bMSF), but decreased that of bovine mammary epithelial cells (bMEC). Proliferation was significantly increased in the bMSF treated with bLF (100μg/mL or greater) as compared with unstimulated cells. The maximal proliferative effect of bLF on bMSF occurred at 1,000μg/mL, such that the proliferation of the bLF-stimulated bMSF was approximately 2.5 times that of unstimulated cells. The bLF increased the production of proliferating cell nuclear antigen and rapid phosphorylation of the p44/p42 mitogen-activated protein kinase in bMSF, but not in bMEC. The bLF-induced proliferation and production of proliferating cell nuclear antigen in bMSF was suppressed by U0126, a specific inhibitor of mitogen-activated protein kinase. Furthermore, treatment with bLF for 24h decreased the mRNA levels of the 3 isoforms of transforming growth factor β in bMSF (16-66%) but upregulated those in bMEC (122-157%). These opposite effects of bLF on the proliferation of epithelial and fibroblast cells and their expression of transforming growth factor β may play a crucial role in bovine mammary involution and remodeling. PMID:25497822

  12. Murine and bovine γδ T cells enhance innate immunity against Brucella abortus infections.

    Directory of Open Access Journals (Sweden)

    Jerod A Skyberg

    Full Text Available γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ(-/- mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα(-/-, and GMCSF(-/- mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ(-/- mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.

  13. Histone modifications and mRNA expression in the inner cell mass and trophectoderm of bovine blastocysts.

    Science.gov (United States)

    Herrmann, Doris; Dahl, John Arne; Lucas-Hahn, Andrea; Collas, Philippe; Niemann, Heiner

    2013-03-01

    Normal development depends on the precise sequence of changes in the configuration of chromatin; these are primarily related to specific biochemical modifications such as acetylation or methylation of histones and DNA methylation. While the role of DNA methylation during preimplantation development has been studied extensively, little is known about histone modifications related to early embryonic development. Here, we investigated gene-specific histone modifications in in vitro produced bovine blastocysts. Selected genes thought to be critical for bovine preimplantation development were examined and included POU5F1 (OCT4), NANOG, INFT, GAPDH, SLC2A3 and IGF1. We used chromatin immunoprecipitation from pools of bovine blastocysts to unravel several modifications of histone H3 in relation to mRNA expression profiles. We focused on the two cell compartments of the blastocyst, the inner cell mass (ICM) and the trophectoderm (TE). We show that gene expression patterns in the ICM and TE of the bovine blastocyst are consistent with histone modification patterns on the promoter of the corresponding genes. The data show a complex epigenetic pattern of promoter occupancy by transcriptionally permissive and repressive H3 modifications. These results pave the way to in-depth epigenetic studies of preimplantation embryos that are crucial to gain a better understanding of the epigenetic changes frequently observed after use of assisted reproductive technologies. PMID:23406883

  14. Isolation of a mutant MDBK cell line resistant to bovine viral diarrhea virus infection due to a block in viral entry.

    Science.gov (United States)

    Flores, E F; Donis, R O

    1995-04-20

    A cell line, termed CRIB, resistant to infection with bovine viral diarrhea virus (BVDV) has been derived from the MDBK bovine kidney cell line. CRIB cells were obtained by selection and cloning of cells surviving infection with a highly cytolytic BVDV strain. CRIB cells contain no detectable infectious or defective BVDV as ascertained by cocultivation, animal inoculation, indirect immunofluorescence, Western immunoblot, Northern hybridization, and RNA PCR. Inoculation of CRIB cells with 24 cytopathic and noncytopathic BVDV strains does not result in expression of viral genes or amplification of input virus. Karyotype and isoenzyme analyses demonstrated that CRIB are genuine bovine cells. CRIB cells are as susceptible as the parental MDBK cells to 10 other bovine viruses, indicating that these cells do not have a broad defect blocking viral replication. Transfection of CRIB cells with BVDV RNA or virus inoculation in the presence of polyethylene-glycol results in productive infection, indicating that the defect of CRIB cells is at the level of virus entry. CRIB cells are the first bovine cells reported to be resistant to BVDV infection in vitro and may be a useful tool for studying the early interactions of pestiviruses with host cells.

  15. Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

    OpenAIRE

    Verdanova, Martina; Pytlik, Robert; Kalbacova, Marie Hubalek

    2014-01-01

    A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate ...

  16. Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

    Science.gov (United States)

    Zhou, M M; Wu, Y M; Liu, H Y; Liu, J X

    2015-04-01

    This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7 μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.

  17. A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

    Institute of Scientific and Technical Information of China (English)

    Hong-yan Guo; Zhi-bin Liang; Yue Li; Juan Tan; Qi-min Chen; Wen-tao Qiao

    2011-01-01

    In order to improve the accuracy for quantitating the bovine foamy virus(BFV)in vitro,we developed a baby hamster kidney cell(BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter(LTR,from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection(MOI)of BFV and the activated ratio of luciferase expression in BFVL. Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.

  18. Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplantation embryos

    DEFF Research Database (Denmark)

    Jensen, Pernille L; Beck, Hans Christian; Petersen, Tonny S;

    2014-01-01

    The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass...... slightly later stage cell differentiation in the developing bovine embryo. In vitro-produced Day 6 embryos were transferred into a recipient heifer and after 7 days of further in vivo culture, ovoid and elongated Day 13 embryos were recovered by flushing both uterine horns after slaughter. The primitive YS...... differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research...

  19. Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells

    Directory of Open Access Journals (Sweden)

    Looft C

    2011-01-01

    Full Text Available Abstract Background The bi-directional communication between the oocyte and its companion cumulus cells (CCs is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa. Results We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO + CCs and without (OO - CCs CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs + OO or without (CCs - OO their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs + OO and CCs - OO, respectively. While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18, translation (EIF2AK1, EIF4ENIF1 and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI, protein metabolic processes (IHH, APOA1, PLOD1, steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7. Similarly, while transcripts over expressed in OO + CCs

  20. Ghrelin stimulates myogenic differentiation in a mouse muscle satellite cell line and in primary cultures of bovine myoblasts.

    Science.gov (United States)

    Montoya-Flores, D; Mora, O; Tamariz, E; González-Dávalos, L; González-Gallardo, A; Antaramian, A; Shimada, A; Varela-Echavarría, A; Romano-Muñoz, J L

    2012-08-01

    Ghrelin is an acylated hormone that influences food intake, energy metabolism and reproduction, among others. Ghrelin may also stimulate proliferating myoblast cell differentiation and multinucleated myotube fusion. The aim of this work was to assess the effect of human ghrelin (hGHRL) and human ghrelin fragment 1-18 (hGHRL1-18) on myoblast differentiation by means of mRNA expression and protein level. Two types of cells were tested, the cell line i28 obtained from mouse skeletal muscle and primary cultures of bovine myoblasts. Both ghrelin and its N-terminal fragment hGHRL1-18 were used at concentrations of 0, 0.01, 0.1, 1, 10 and 100 nm. Treatments were applied to pre-confluent cultures and were maintained for 4 days. We determined that between 0.1 and 100 nm, hGHRL and hGRHL1-18 had similar effects on myogenic differentiation of i28 cells (p < 0.01). On the other hand, only the higher concentrations (10 and 100 nm) of hGHRL stimulated bovine myoblast differentiation. These results could be attributed to the presence, in both i28 cells and in bovine myoblasts, of the mRNA for GHS-R1a and CD36 receptors. The use of ghrelin in livestock production is still questionable because of the limited effects shown in this study, and additional research is needed in this field. PMID:21777295

  1. Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine innate immune cells

    Directory of Open Access Journals (Sweden)

    Pirson Chris

    2012-06-01

    Full Text Available Abstract Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM and trehalose monomycolate (TMM, the apolar phthiocerol dimycocersates (PDIMs, triacyl glycerol (TAG, pentacyl trehalose (PAT, phenolic glycolipid (PGL, and mono-mycolyl glycerol (MMG. Polar lipids identified included glucose monomycolate (GMM, diphosphatidyl glycerol (DPG, phenylethanolamine (PE and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs. These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.

  2. In vitro effects of the organochlorine pesticide β-hexachlorocyclohexane on bovine peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Cristina Rossi

    2014-09-01

    Full Text Available The β-hexachlorocyclohexane (β-HCH is a very stable and accumulable isomer of Lindane, a well known organochlorine pesticide. The HCHs were banned in all developed countries but to date high concern still exists for environment, animal and human health due to contaminated sites. In this study, several in vitro tests [cell viability (XTT, trypan blue exclusion (TBE, lactate dehydrogenase release (LDH and bromodeoxyuridine (BrdU incorporation assays] were performed to investigate the toxic effects of exposure to β-HCH (from 0.1 to 1000 μM on bovine peripheral blood mononuclear cells (PBMCs. All the trials were performed incubating PBMCs for 2 and 7 days. At high concentrations (i.e. 1000 μM, the β-HCH approximately halved the number of living cells regardless the exposure time, significantly decreased the cell viability assessed by the XTT assay, and compromised the proliferation potential of PBMCs. At lower β-HCH exposure levels (0.1 to 100 μM, particularly after 7 days of exposure, a progressive decrease of cell viability has been observed. These adverse effects were significant at concentrations observed in the blood of cattle reared in polluted areas. The LDH results suggest that β-HCH does not clearly affect the integrity of the cell membrane in the range of exposure levels tested. All in all, these findings warn about the risk posed by the long-term exposure to β-HCH of farm animals reared in rural areas polluted by β-HCH. Further research is needed to deepen our knowledge about the mechanisms through which β-HCH affects the PBMCs functionality.

  3. Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture

    OpenAIRE

    Klagsbrun, M

    1980-01-01

    Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco’s modified eagle’s medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow...

  4. The Protozoan Neospora caninum Directly Triggers Bovine NK Cells To Produce Gamma Interferon and To Kill Infected Fibroblasts

    Science.gov (United States)

    Boysen, Preben; Klevar, Siv; Olsen, Ingrid; Storset, Anne K.

    2006-01-01

    Natural killer (NK) cells are considered to be key players in the early innate responses to protozoan infections, primarily indirectly by producing gamma interferon (IFN-γ) in response to cytokines, like interleukin 12 (IL-12). We demonstrate that live, as well as heat-inactivated, tachyzoites of Neospora caninum, a Toxoplasma-like protozoan, directly trigger production of IFN-γ from purified, IL-2-activated bovine NK cells. This response occurred independently of IL-12 but was increased by the addition of the cytokine. A similar IFN-γ response was measured in cocultures of NK cells and N. caninum-infected autologous fibroblasts. However, no NK cell-derived IFN-γ response was detected when cells were cultured with soluble antigens from the organism, indicating that intact tachyzoites or nonsoluble components are necessary for NK cell triggering. Furthermore, N. caninum-infected autologous fibroblasts had increased susceptibility to NK cell cytotoxicity compared to uninfected fibroblasts. This cytotoxicity was largely mediated by a perforin-mediated mechanism. The activating receptor NKp46 was involved in cytotoxicity against fibroblasts but could not explain the increased cytotoxicity against infected targets. Interestingly, N. caninum tachyzoites were able to infect cultured NK cells, in which tachyzoites proliferated inside parasitophorous vacuoles. Together, these findings underscore the role of NK cells as primary responders during a protozoan infection, describe intracellular protozoan infection of NK cells in vitro for the first time, and represent the first functional study of purified bovine NK cells in response to infection. PMID:16428740

  5. Quantitation of cytokine gene expression by real time PCR in bovine milk and colostrum cells from cows immunized with a bovine rotavirus VP6 experimental vaccine.

    Science.gov (United States)

    Gonzalez, D D; Rimondi, A; Perez Aguirreburualde, M S; Mozgovoj, M; Bellido, D; Wigdorovitz, A; Dus Santos, M J

    2013-10-01

    In a previous work, VP6 recombinant protein was produced using baculovirus system and it was evaluated in a colostrum-deprived calf model. This vaccine was able to protect calves against viral challenge without inducing neutralizing antibodies (NAb), suggesting that another immunological effectors were involved in the protection observed. In this work, groups of cows (n=4) were immunized in the last third of gestation with a bovine rotavirus (BRV) experimental vaccine and with a VP6 subunit vaccine. At birth, colostrums from vaccinated and non-vaccinated cows were processed and viable colostral mononuclear cells were obtained. With the purpose of determining the cytokine patterns generated by cells from immune secretions (colostrums and milk), a relative quantification by real time PCR was standardized. Quantitative real time PCR (qPCR) was used to determine transcript levels of IL-4, IL-6, IL-10, IL-12, IFN-γ and IFN-α from these cells. Colostral and milk mononuclear cells expressed a different cytokine transcript expression pattern regarding the vaccine used. These results demonstrated that the colostral cellular population was active and could exert its action influencing the final immune response. PMID:23602433

  6. DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs

    Directory of Open Access Journals (Sweden)

    Beheshteh Abouhamzeh

    2015-01-01

    Full Text Available Objective: Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer (SCNT. We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells (BADSCs would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b and histone deacetyltransferses (HDAC1, HDAC2, HDAC3 in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression (OCT4 and acetylation of H3K9 (H3K9ac in BADSCs cultures and different passages in vitro. Materials and Methods: In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction (q-PCR, and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 (P3, 5 (P5 and 7 (P7. Results: The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Conclusion: Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages.

  7. Expression and localization of inwardly rectifying potassium channel Kit2.1 in glia cells of native bovine retina

    Institute of Scientific and Technical Information of China (English)

    PAN Ai-hua; LUO Xue-gang

    2005-01-01

    Objective The purpose of this study is to identify the molecular basis of the contacting -neuron membrane K+ conductance in glia cells of native bovine retina. Methods RT-PCR, Northern blot and Western blot analyses were used to detect the expression of the inwardly rectifying K+ (Kir) channel subunits Kir2.1 in native bovine RPE and neural retina. The distribution of Kir2.1 protein was determined in frozen sections of bovine retina-RPEchoroid by indirect immunofluorescence analysis. Results RT-PCR analysis reveals Kir2.1 transcript in both RPE and neural retina. In Northern blots, Kir2.1 probe hybridizes to an appropriately sized-transcript in neural retina but not in RPE. In Western blots, Kir2.1 antibody recognizes a major monomer of about 60 kDa in neural retina but not in RPE. Immunofluorescence reveals that Kir2.1 immunostaining is expressed at many parts of Muller cells, especially in the membrane domains of Muller cells that contact retinal neurons, i. e. , along the two stem processes,over the soma, and in the side branches extending into the synaptic layers. No immunostaining is seen in RPE. Doubling staining shows that Kir2.1 proteins and glutamine synthetase proteins which are a marker of Muller cell co-localized well. Conclusions These results reveal that Kir2.1 is localized in the Muller cells, no Kir2.1 in RPE. These data suggests that Kir2.1 may be involved in the transport of K+ in the bovine neural retina.

  8. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  9. Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

    Science.gov (United States)

    Wang, H-M; Xia, X-Z; Hu, G-X; Yu, L; He, H-B

    2016-03-01

    Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA. PMID:26982472

  10. The influence of protein fractions from bovine colostrum digested in vivo and in vitro on human intestinal epithelial cell proliferation.

    Science.gov (United States)

    Morgan, Alison J; Riley, Lisa G; Sheehy, Paul A; Wynn, Peter C

    2014-02-01

    Colostrum consists of a number of biologically active proteins and peptides that influence physiological function and development of a neonate. The present study investigated the biological activity of peptides released from first day bovine colostrum through in vitro and in vivo enzymatic digestion. This was assessed for proliferative activity using a human intestinal epithelial cell line, T84. Digestion of the protein fraction of bovine colostrum in vitro was conducted with the enzymes pepsin, chymosin and trypsin. Pepsin and chymosin digests yielded protein fractions with proliferative activity similar to that observed with undigested colostrum and the positive control foetal calf serum (FCS). In contrast trypsin digestion significantly (P<0·05) decreased colostral proliferative activity when co-cultured with cells when compared with undigested colostrum. The proliferative activity of undigested colostrum protein and abomasal whey protein digesta significantly increased (P<0·05) epithelial cell proliferation in comparison to a synthetic peptide mix. Bovine colostrum protein digested in vivo was collected from different regions of the gastrointestinal tract (GIT) in newborn calves fed either once (n=3 calves) or three times at 12-h intervals (n=3 calves). Digesta collected from the distal duodenum, jejunum and colon of calves fed once, significantly (P<0·05) stimulated cell proliferation in comparison with comparable samples collected from calves fed multiple times. These peptide enriched fractions are likely to yield candidate peptides with potential application for gastrointestinal repair in mammalian species. PMID:24433585

  11. PECAM-1 is involved in neutrophil transmigration across Histophilus somni treated bovine brain endothelial cells.

    Science.gov (United States)

    Tiwari, Raksha; Sullivan, J; Czuprynski, C J

    2009-09-01

    Histophilus somni (H. somni) is a gram-negative bacterial pathogen that causes respiratory, reproductive, and central nervous system disease in cattle. The hallmark of systemic H. somni infection is diffused vasculitis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because platelet endothelial cell adhesion molecule-1 (PECAM-1) and endothelial nitric oxide synthase (eNOS) play fundamental roles in maintaining homeostasis in blood vessels, we sought to determine if PECAM-1 and eNOS expression play a role in events related to the pathogenesis of TME. Our findings demonstrate that neutrophil transmigration across H. somni-treated TBBEC (SV-40 transformed bovine brain endothelial cell line) was reduced by treatment with anti-PECAM-1 antibodies. Confocal microscopy indicated that H. somni treatment leads to redistribution of PECAM-1 and eNOS on the surface of TBBEC. These findings suggest that PECAM-1 and eNOS may play a role in the early pathogenesis of TME. PMID:19524660

  12. Making the Switch: Alternatives to Fetal Bovine Serum for Adipose-Derived Stromal Cell Expansion

    Science.gov (United States)

    Dessels, Carla; Potgieter, Marnie; Pepper, Michael S.

    2016-01-01

    Adipose-derived stromal cells (ASCs) are being used extensively in clinical trials. These trials require that ASCs are prepared using good manufacturing practices (GMPs) and are safe for use in humans. The majority of clinical trials in which ASCs are expanded make use of fetal bovine serum (FBS). While FBS is used traditionally in the research setting for in vitro expansion, it does carry the risk of xenoimmunization and zoonotic transmission when used for expanding cells destined for therapeutic purposes. In order to ensure a GMP quality product for cellular therapy, in vitro expansion of ASCs has been undertaken using xeno-free (XF), chemically-defined, and human blood-derived alternatives. These investigations usually include the criteria proposed by the International Society of Cellular Therapy (ISCT) and International Fat Applied Technology Society (IFATS). The majority of studies use these criteria to compare plastic-adherence, morphology, the immunophenotype and the trilineage differentiation of ASCs under the different medium supplemented conditions. Based on these studies, all of the alternatives to FBS seem to be suitable replacements; however, each has its own advantages and drawbacks. Very few studies have investigated the effects of the supplements on the immunomodulation of ASCs; the transcriptome, proteome and secretome; and the ultimate effects in appropriate animal models. The selection of medium supplementation will depend on the downstream application of the ASCs and their efficacy and safety in preclinical studies. PMID:27800478

  13. Effects of mycotoxins in cultured kidney cells: cytotoxicity of aflatoxin B1 in Madin-Darby and primary fetal bovine kidney cells.

    Science.gov (United States)

    Yoneyama, M; Sharma, R P; Elsner, Y Y

    1987-04-01

    The cytotoxicity of aflatoxin B1, a fungal metabolite and an important food contaminant, was evaluated in an established cell line, Madin-Darby bovine kidney (MDBK) cells, and in primary fetal bovine kidney (PFBK) cells. Cells were grown in monolayers and treated with media containing AFB1 for 24 hr. Both culture systems were sensitive to the chemical; the PFBK cultures were approximately four times more susceptible to the cytotoxic effect. Cell multiplication decreased in both systems in long-term cultures. Adherence of MDBK cells was only slightly reduced at the toxic concentrations of the chemical. Electron microscopy revealed condensation of chromatin, separation of nuclei from the cytoplasm, cytoplasmic vacuolization, and loss of surface microvilli. Results indicated differential sensitivity of the two culture systems.

  14. Quantitative analysis of infiltrating immune cells and bovine papillomavirus type 1 E2-positive cells in equine sarcoids.

    Science.gov (United States)

    Geisshüsler, H; Marti, E; Stoffel, M H; Kühni, K; Stojiljkovic, A; von Tscharner, C; Vidondo, B; Gerber, V; Koch, C

    2016-10-01

    Sarcoids are the most frequently observed skin tumours in equids and consist of cutaneous accumulations of transformed fibroblasts. Their aetiopathogenesis is closely linked to a presumably abortive infection by bovine papillomavirus (BPV) types 1 and 2. In cattle, dermal fibropapillomas induced by BPV1/2 usually regress spontaneously due to a local, cell-mediated, immune response; however, equids appear to lack an effective immune response to BPV1/2 and mechanisms of immune evasion have been postulated. As a consequence, equine sarcoids tend to persist and are prone to recur. In this study, cryosections were analysed by immunofluorescent staining and a high content analysis system to determine the presence and distribution of CD4(+), CD8(+), FoxP3(+), RORγt(-), CD206(+) and CD14(+) cells, along with expression of the BPV1 early regulatory protein E2. A higher density of cells was positive for BPV1 E2(+) within the transformed tissue than in perilesional tissue or normal skin of horses with sarcoids and control horses. The proportion of CD8(+) cytotoxic T cells was significantly increased in perilesional and lesional tissues, whereas CD4(+) T helper cells were present in higher density only in lesional tissue compared to normal skin from horses with and without sarcoids. The proportion of pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells was decreased in sarcoid tissue compared to perilesional, distant and control tissue. There were no significant differences in densities of CD4(+)FoxP3(+) RORγt(-) regulatory T cells between sarcoids and control tissues. Equine sarcoids are characterised by infiltrations of CD8(+) and CD4(+) T cells, with decreased representation by pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells. PMID:27687925

  15. Mesenchymal stem cells from amnion and amniotic fluid in the bovine.

    Science.gov (United States)

    Corradetti, B; Meucci, A; Bizzaro, D; Cremonesi, F; Lange Consiglio, A

    2013-04-01

    Amnion and amniotic fluid (AF) are noncontroversial and inexhaustible sources of mesenchymal stem cells (MSCs) that can be harvested noninvasively at low cost. As in humans, also in veterinary field, presumptive stem cells derived from these tissues reveal as promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. The aim of this work is to obtain and characterize, for the first time in bovine species, presumptive MSCs from the epithelial portion of the amnion (AECs) and from the AF (AF-MSCs) to be used for clinical applications. AECs display a polygonal morphology, whereas AF-MSCs exhibit a fibroblastic-like morphology only starting from the second passage, being heterogeneous during the primary culture. For both lines, the proliferative ability has been found constant over the ten passages studied and AECs show a statistically lower (P<0.05) doubling time with respect to AF-MSCs. AECs express MSC-specific markers (ITGB1 (CD29), CD44, ALCAM (CD166), ENG (CD105), and NT5E (CD73)) from P1 to P3; in AF-MSCs, only ITGB1, CD44, and ALCAM mRNAs are detected; NT5E is expressed from P2 and ENG has not been found at any passage. AF-MSCs and AECs are positive for the pluripotent markers (POU5F1 (OCT4) and MYC (c-Myc)) and lack of the hematopoietic markers. When appropriately induced, both cell lines are capable of differentiating into ectodermal and mesodermal lineages. This study contributes to reinforce the emerging importance of these cells as ideal tools in veterinary medicine. A deeper evaluation of the immunological properties needs to be performed in order to better understand their role in cellular therapy.

  16. Generation of lipid neutrophil chemoattractant by irradiated bovine aortic endothelial cells.

    Science.gov (United States)

    Matzner, Y; Cohn, M; Hyam, E; Razin, E; Fuks, Z; Buchanan, M R; Haas, T A; Vlodavsky, I; Eldor, A

    1988-04-15

    Radiation injury to blood vessels is associated with an acute inflammatory process. We investigated the capacity of cultured bovine aortic endothelial cells (BAEC) to produce chemotactic factors after radiation injury. BAEC in serum-free media were irradiated with a cobalt-60 Gammacell 220 and the cell supernatants were assayed for chemotactic activity for human neutrophils in a Boyden chamber. There was a rapid release of chemotactic activity into the BAEC supernatants which was dependent both on the dose of radiation (5 to 40 Gy) and the time between irradiation and sample collection. In contrast, isolation of BAEC lysates by freeze-thawing was not associated with the presence of similar chemotactic activity. The chemotactic activity released from the irradiated BAEC was not destroyed by boiling nor by treatment with trypsin. The release of the chemotactic activity was, however, inhibited by the addition of a lipoxygenase inhibitor but not by the addition of a cyclooxygenase inhibitor before the irradiation. The chemotactic activity was recovered from the cell supernatants in the lipid phase after extraction with chloroform/methanol. Furthermore, the chloroform/methanol extracts co-eluted with authentic leukotriene B4 when the BAEC were prelabeled with [14C] arachidonic acid. However, we were unable to detect endogenous leukotriene B4 with RIA. Instead, the only detectable endogenous lipid present in the supernatants was 13-hydroxyoctadecadienoic acid which is derived from linoleic acid via the lipoxygenase pathway. 13-Hydroxyoctadecadienoic acid, however, had no chemotactic activity. These findings suggest that endothelial cells rapidly release a chemotactic agent after irradiation, the release of which is associated with a lipoxygenase pathway. The release of this chemotactic activity may account in part for the acute inflammatory response that is observed after ionizing irradiation.

  17. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  18. DNA methylation status of H19 and Xist genos in lungs of somatic cell nuclear transfer bovines

    Institute of Scientific and Technical Information of China (English)

    CHEN Jie; LI DongJie; LIU YanQin; ZHANG Cui; DAI YunPing; LI ShiJie; LINing

    2008-01-01

    In somatic cell nuclear transfer (SCNT) technologies, the donor cell's nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epige- netic modification of the genome that regulates crucial aspects of genome function, including estab-lishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals, we analyzed the DNA methylation status of two imprinting genes, H19 and Xist, in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05), and three tested CpGs sites (1, 2, 3) exhibited unmethylation in one cloned bovine (9C3); however, Xist showed similar DNA methylation levels between clones and con- trols, and both showed hypermethylation (96.11% and 86.67%).

  19. Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (20 C)

    Science.gov (United States)

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2009-01-01

    Summary Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 C/min in a low-temperature (80 C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; 20 C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type. ?? 2009 Cambridge University Press.

  20. Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

    Science.gov (United States)

    Headley, Colwyn A; DiSilvestro, David; Bryant, Kelsey E; Hemann, Craig; Chen, Chun-An; Das, Amlan; Ziouzenkova, Ouliana; Durand, Grégory; Villamena, Frederick A

    2016-03-15

    Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

  1. Bovine colostrum supports the serum-free proliferation of epithelial cells but not of fibroblasts in long-term culture

    Science.gov (United States)

    Klagsbrun, M

    1980-01-01

    Medium lacking serum but supplemented with milk will support the growth of sparse cells in culture. Milk obtained within 8 h after the birth of a calf (day 1 colostrum) is the most effective in supporting proliferation. In mixed cultures of early-passage bovine embryonic kidney (BEK) or early-passage calf kidney (CK) cells, both epithelial cells and fibroblasts grow in Dulbecco’s modified eagle’s medium (DMEM) supplemented with serum. However, only cells that appear to be epithelial-like grow in DMEM supplemented with colostrum. Sparse cultures of early-passage human and rat fibroblasts that grow readily in DMEM supplemented with serum do not grow in DMEM supplemented with colostrum. Canine kidney epithelial cells (MDCK), when plated sparsely, grow exponentially in DMEM supplemented with day 1 bovine colostrum. The generation time is 26 h, the same growth rate as in DMEM supplemented with calf serum. The MDCK cells can be subcultured and regrown to confluence repeatedly in colostrum-supplemented DMEM. Growth in DMEM supplemented with colostrum does not alter the morphological characteristics of the MDCK cells, which are polygonal, contain microvilli at the apical surface, and are connected by tight junctions and desmosomes. MDCK cells do not proliferate in DMEM supplemented with milk obtained 1 wk after the birth of a calf. PMID:7358799

  2. [Morphological observation of bovine kidney (MDBK) cells effected by foot-and-mouth disease virus L(pro)].

    Science.gov (United States)

    Hao, Fengqiang; Cong, Guozheng; Gao, Shandian; Lin, Tong; Du, Junzheng; Shao, Junjun; Chang, Huiyun

    2009-11-01

    In order to explore the morphological changes of Bovine Kidney (MDBK) cells induced by foot-and-mouth disease virus (FMDV) L protease, we induced the expression of FMDV L protease in bovine kidney cells (MDBK) artificially. All work is carried out on the basis of a stable MDBK cell line inducibly expresses the Lab gene under the control of tetracycline. We use cell morphology, Hoechst 33258 staining, AO-EB staining, and DNA Ladder abstraction to research the morphological changes of MDBK cells. 24 hours after FMDV L protease were induced and expressed in MDBK cells, cells shown the diminish of cell size, nuclear enrichment and the appearance of transparency circle under the light microscope. Apoptosis characteristics of nuclear condensation, fragmentation, accompanied by apoptotic bodies formation (Hoechst 33258 staining). 36 hours after the expression, nuclear staining of early lesions showed bright green plaque or debris-like dense, and advanced lesions showed Orange and dense plaques (AO-EB staining). 48 hours after the expression, DNA gel electrophoresis showed visible DNA ladder. Results indicate that FMDV L protease can induce apoptosis of MDBK and apoptosis plays an important role in the cytopathogenicity effect of FMDV.

  3. Dietary bovine lactoferrin alters mucosal and systemic immune cell responses in neonatal piglets.

    Science.gov (United States)

    Comstock, Sarah S; Reznikov, Elizabeth A; Contractor, Nikhat; Donovan, Sharon M

    2014-04-01

    Lactoferrin (LF) is a multifunctional immune protein found at high concentrations in human milk. Herein, the effect of dietary bovine LF (bLF) on mucosal and systemic immune development was investigated. Colostrum-deprived piglets were fed formula containing 130 [control (Ctrl)], 367 (LF1), or 1300 (LF3) mg of bLF/(kg body weight · d). To provide passive immunity, sow serum was provided orally during the first 36 h of life. Blood, spleen, mesenteric lymph node (MLN), and ascending colon (Asc) contents were collected on day 7 (n = 10-14/group) and day 14 (n = 10-12/group). Immune cell populations were quantified by flow cytometry and immunoglobulins (Igs) were measured by ELISA. Additionally, immune cells were isolated from spleen and MLNs (n = 7/group) on day 7 and stimulated ex vivo with phytohemagglutinin or lipopolysaccharide (LPS) ± LF for 72 h. Secreted cytokine concentrations were quantified by multiplex assay. Lymphocyte populations [cluster determinant (CD)4, CD8, and natural killer cells] developed normally and were unaffected by dietary bLF. LF3 piglets tended to have 1.4 to 2 times more serum IgG than Ctrl piglets (P = 0.07) or LF1 piglets (P = 0.03), but IgA in Asc contents was unaffected by bLF. Asc IgA was 4 times higher on day 14 than day 7. Spleen cells from LF3 piglets produced 2 times more interleukin (IL)-10 and tumor necrosis factor (TNF)-α ex vivo than those from Ctrl or LF1 piglets. MLN cells from LF1 and LF3 piglets produced 40% more IL-10 and tended to produce 40% more IL-6 (P = 0.05) than those from Ctrl piglets. However, ex vivo bLF did not affect the cytokine response of spleen or MLN cells to LPS. In summary, dietary bLF alters the capacity of MLN and spleen immune cells to respond to stimulation, supporting a role for LF in the initiation of protective immune responses in these immunologically challenged neonates. PMID:24553692

  4. Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

    Science.gov (United States)

    Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

    2014-07-01

    The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. PMID:24906928

  5. Bovine Induced Pluripotent Stem Cells Are More Resistant to Apoptosis than Testicular Cells in Response to Mono-(2-ethylhexyl Phthalate

    Directory of Open Access Journals (Sweden)

    Ying-Chu Lin

    2014-03-01

    Full Text Available Although the androgen receptor (AR has been implicated in the promotion of apoptosis in testicular cells (TSCs, the molecular pathway underlying AR-mediated apoptosis and its sensitivity to environmental hormones in TSCs and induced pluripotent stem cells (iPSCs remain unclear. We generated the iPSCs from bovine TSCs via the electroporation of OCT4. The established iPSCs were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4 to maintain and stabilize the expression of stemness genes and their pluripotency. Apoptosis signaling was assessed after exposure to mono-(2-ethylhexyl phthalate (MEHP, the active metabolite of di-(2-ethylhexyl phthalate. Here, we report that iPSCs were more resistant to MEHP-induced apoptosis than were original TSCs. MEHP also repressed the expression of AR and inactivated WNT signaling, and then led to the commitment of cells to apoptosis via the cyclin dependent kinase inhibitor p21CIP1. The loss of the frizzed receptor 7 and the gain of p21CIP were responsible for the stimulatory effect of MEHP on AR-mediated apoptosis. Our results suggest that testicular iPSCs can be used to study the signaling pathways involved in the response to environmental disruptors, and to assess the toxicity of environmental endocrine disruptors in terms of the maintenance of stemness and pluripotency.

  6. Cytopathic BVDV-1 strain induces immune marker production in bovine cells through the NF-κB signaling pathway.

    Science.gov (United States)

    Fredericksen, Fernanda; Carrasco, Gonzalo; Villalba, Melina; Olavarría, Víctor H

    2015-12-01

    The bovine viral diarrhea virus (BVDV-1) is a pathogen responsible for high economic losses in the cattle industry worldwide. This virus has the capacity to modulate the immune system of several higher vertebrates, but there is little information available on the cell infection mechanism. To further investigate the effects of BVDV-1 on the activation of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the proinflammatory and antiviral cytokine expression profiles were analyzed. The results showed that BVDV-1 was able to induce the production of BCL3, IL-1β, IL-8, IL-15, IL-18, Mx-1, IRF-1, and IRF-7 in a way similar to polyinosinic-polycytidylic acid. Interestingly, all BVDV-1 activities were blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that BVDV-1 regulates gene expression in bovines through the activation of several key transcription factors. Collectively, these data identified BVDV-1 as a viral regulator of immune marker expression, even from early infection. Additionally, this is the first report to find BVDV-1 modulating the activation of cytokine production and transcriptions factors mainly through the NF-κB pathway in vertebrates. PMID:26330089

  7. Adrenaline-, noradrenaline- and small granule- containing cells in the adrenal gland of Discoglossus pictus (Amphibia, Anura).

    OpenAIRE

    Accordi, F; Gallo, V P

    1982-01-01

    In the adrenal gland of Discoglossus pictus, various types of chromaffin cells are described: noradrenaline cells, adrenaline cells and small granule-containing cells (on the basis of electron density and shape of the granules). The chromaffin cells occur in small groups, and have cytoplasmic processes which may surround them in the form of parallel layers. Their nerve supply is sparse. The possible function of SGC-cells, in relation to those described in other vertebrates, is discussed.

  8. Bovine neonatal pancytopenia - Comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK

    Directory of Open Access Journals (Sweden)

    Euler Kerstin N

    2013-01-01

    Full Text Available Abstract Background Bovine neonatal pancytopenia (BNP is a disease syndrome in newborn calves of up to four weeks of age, first observed in southern Germany in 2006. By now, cases have been reported in several countries around the globe. Many affected calves die within days due to multiple haemorrhages, thrombocytopenia, leukocytopenia and bone marrow depletion. A certain vaccine directed against Bovine Virus Diarrhoea Virus (BVDV was recently shown to be associated with BNP pathogenesis. Immunized cows develop alloantibodies that are transferred to newborn calves via colostrum intake. In order to further elucidate BNP pathogenesis, the purpose of this study was to characterize and compare the protein composition of the associated vaccine to another vaccine directed against BVDV not related to BNP and the cell surface proteome of MDBK (Madin-Darby Bovine Kidney cells, the cell line used for production of the associated vaccine. Results By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production. Conclusions The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.

  9. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    Science.gov (United States)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ -1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non

  10. β-Hydroxybutyrate Facilitates Fatty Acids Synthesis Mediated by Sterol Regulatory Element-Binding Protein1 in Bovine Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Min Zhang

    2015-11-01

    Full Text Available Background/Aims: In dairy cows, β-hydroxybutyrate (BHBA is utilized as precursors of de novo synthesized fatty acids in mammary gland. Ketotic cows are characterized by excessive negative energy balance (NEB, which can further increase the blood BHBA concentration. Sterol regulatory element-binding protein1 (SREBP1 and cell death-inducing DNA fragmentation factor-alpha-like effector α (Cidea play crucial roles in lipid synthesis. Therefore, we hypothesized that BHBA could stimulate SREBP1/Cidea pathway to increase milk fat synthesis in bovine mammary epithelial cells. Methods: Bovine mammary epithelial cells were treated with different concentrations of BHBA and transfected with adenovirus to silence SREBP1 expression. The effects of BHBA on the lipid synthesis in bovine mammary epithelial cells were investigated. Results: The results showed that BHBA could significantly increase the expression of SREBP1, fatty acid synthase (FAS, acetyl-CoA carboxylase α (ACC-α, Cidea and diacylglycerol transferase-1 (DGAT-1, as well as the triglycerides (TG content in bovine mammary epithelial cells. BHBA treatment also increased the transfer of mature SREBP1 to nucleus compared with control group. However, SREBP1 silencing could significantly down-regulate the overexpression of FAS, ACC-α, Cidea and DGAT-1, as well as TG content induced by BHBA. Conclusion: The present data indicate that BHBA can significantly increase TG secretion mediated by SREBP1 in bovine mammary epithelial cells.

  11. Anti-Müllerian hormone (AMH) receptor type II expression and AMH activity in bovine granulosa cells.

    Science.gov (United States)

    Poole, Daniel H; Ocón-Grove, Olga M; Johnson, Alan L

    2016-09-15

    Anti-Müllerian hormone (AMH) produced by granulosa cells has previously been proposed to play a role in regulating granulosa cell differentiation and follicle selection. Although AMH receptor type II (AMHR2) dimerizes with a type I receptor to initiate AMH signaling, little is known about the regulation of AMHR2 expression in bovine granulosa cells and the role of AMH in follicle development. The primary objectives of this study were to: (1) characterize AMHR2 expression in granulosa cells during follicle development; (2) identify factors that regulate AMHR2 mRNA expression in granulosa cells; and (3) examine the role of AMH signaling in granulosa cell differentiation and proliferation. Bovine granulosa cells were isolated from 5- to 8-mm follicles before selection and deviation, as well as from 9- to 12-mm and 13- to 24-mm follicles after selection. Analyses revealed that expression of AMHR2 was greater in 5- to 8-mm follicles compared with 13- to 24-mm follicles (P AMH was greater in granulosa cells cultured with BMP2, BMP6, or BMP15 when compared with controls (P AMH, in vitro, inhibited CYP19A1 expression in a dose-related (10-100 ng/mL) fashion, and reduced granulosa cell proliferation at 48 and 72 hours (P AMH signaling plays a role in both regulating granulosa cell proliferation and preventing granulosa cells from 5- to 8-mm follicles from undergoing premature differentiation before follicle selection. PMID:27268296

  12. Genome sequence of foot-and-mouth disease virus outside the 3A region is also responsible for virus replication in bovine cells.

    Science.gov (United States)

    Ma, Xueqing; Li, Pinghua; Sun, Pu; Lu, Zengjun; Bao, Huifang; Bai, Xingwen; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

    2016-07-15

    The deletion of residues 93-102 in non-structure protein 3A of foot-and-mouth disease virus (FMDV) is associated with the inability of FMDV to grow in bovine cells and attenuated virulence in cattle.Whereas, a previously reported FMDV strain O/HKN/21/70 harboring 93-102 deletion in 3A protein grew equally well in bovine and swine cells. This suggests that changes inFMDV genome sequence, in addition to 93-102 deletion in 3A, may also affectthe viral growth phenotype in bovine cellsduring infection and replication.However, it is nuclear that changes in which region (inside or outside of 3A region) influences FMDV growth phenotype in bovine cells.In this study, to determine the region in FMDV genomeaffecting viral growth phenotype in bovine cells, we constructed chimeric FMDVs, rvGZSB-HKN3A and rvHN-HKN3A, by introducing the 3A coding region of O/HKN/21/70 into the context of O/SEA/Mya-98 strain O/GZSB/2011 and O Cathay topotype strain O/HN/CHA/93, respectively, since O/GZSB/2011 containing full-length 3A protein replicated well in bovine and swine cells, and O/HN/CHA/93 harboring 93-102 deletion in 3A protein grew poorly in bovine cells.The chimeric virusesrvGZSB-HKN3A and rvHN-HKN3A displayed growth properties and plaque phenotypes similar to those of the parental virus rvGZSB and rv-HN in BHK-21 and primary fetal porcine kidney (FPK) cells. However, rvHN-HKN3A and rv-HN replicated poorly in primary fetal bovine kidney (FBK) cells with no visible plaques, and rvGZSB-HKN3A exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the difference present in FMDV genome sequence outside the 3A coding region also have influence on FMDV replication ability in bovine cells. PMID:27094491

  13. Cell viability of bovine spermatozoa subjected to DNA electroporation and DNAse I treatment.

    Science.gov (United States)

    Cavalcanti, Paulo Varoni; Milazzotto, Marcella Pecora; Simões, Renata; Nichi, Marcilio; de Oliveira Barros, Flavia Regina; Visintin, Jose Antonio; Assumpção, Mayra Elena Ortiz D'Avila

    2016-04-15

    Many mechanisms involved in sperm-mediated gene transfer (SMGT) are still unknown. It is still a matter of debate whether exogenous DNA fragments incorporated by the embryo are originated from those bound to the sperm membrane or by those that penetrated the intracellular compartment. In an attempt to elucidate the transmission mechanism of exogenous DNA molecules by sperm, some authors suggested a treatment with DNAse I to remove DNA molecules outside the sperm. But little is known regarding the effects of DNAse I treatment on sperm viability and its impact on sperm organelles. An important aspect of the SMGT technique is the amount of exogenous DNA incubated with sperm, which may influence the internalization rate. Due to the inconsistencies found in literature, this work aimed to contribute to bovine sperm physiology knowledge evaluating the effects of different DNA concentrations, electroporation, and DNAse I treatments on sperm viability characteristics, DNA uptake, and IVF. For that, the effects of different concentrations of exogenous DNA (250, 500 and 1000 ng/10(6) cells) and incubation or electroporation were tested on sperm functional characteristics and in vitro embryo production. No effect of DNA concentration was observed on uptake, plasma membrane integrity, and mitochondrial membrane potential. The addition of exogenous DNA induced a decrease on acrosomal lesion in the 500-ng group when compared to the control. Cells incubated with DNA, electroporated, and treated with DNAse I presented a deleterious influence on mitochondrial membrane potential. In vitro fertilization was made with 1000 ng of DNA, sperm cells incubated or electroporated followed by DNAse I treatment. No significant difference was found in cleavage rate. Blastocyst rates were 24.36% for the control; 19.65% for incubated; 3.5% for electroporated control; and 17.40% for electroporated. There is a significant difference in blastocyst rate between the control and electroporated

  14. A gene expression programme induced by bovine colostrum whey promotes growth and wound-healing processes in intestinal epithelial cells.

    Science.gov (United States)

    Blais, M; Pouliot, Y; Gauthier, S; Boutin, Y; Lessard, M

    2014-01-01

    Bovine colostrum is well known for its beneficial properties on health and development. It contains a wide variety of bioactive ingredients that are known to promote a number of cellular processes. Therefore the use of colostrum whey as a feed additive to promote intestinal health has been proposed, yet little is known about mechanisms implicated in its beneficial properties on intestinal epithelial cells. In the present paper, casein were removed from bovine colostrum and the remaining liquid, rich in bioactive compounds, was evaluated for its capacity to modulate cellular processes in porcine intestinal epithelial cell line IPEC-J2 and human colon adenocarcinoma cell line Caco-2/15. First, we verified the effect of colostrum whey and cheese whey on processes involved in intestinal wound healing, including cell proliferation, attachment, morphology and migration. Our results showed that colostrum whey promoted proliferation and migration, and decreased specifically the attachment of Caco-2/15 cells on the culture dish. On the other hand, cheese whey induced proliferation and morphological changes in IPEC-J2 cells, but failed to induce migration. The gene expression profile of IPEC-J2 cells following colostrum whey treatment was evaluated by microarray analysis. Results revealed that the expression of a significant number of genes involved in cell migration, adhesion and proliferation was indeed affected in colostrum whey-treated cells. In conclusion, colostrum specific bioactive content could be beneficial for intestinal epithelial cell homoeostasis by controlling biological processes implicated in wound healing through a precise gene expression programme. PMID:26101625

  15. Comparative Analysis of the miRNome of Bovine Milk Fat, Whey and Cells

    Science.gov (United States)

    Li, Ran; Dudemaine, Pier-Luc; Zhao, Xin; Lei, Chuzhao; Ibeagha-Awemu, Eveline Mengwi

    2016-01-01

    RNA activities in bovine mammary gland. Predicted target genes (1802) of 14 highly expressed miRNAs in milk fractions were enriched in fundamental cellular functions, infection, organ and tissue development. Furthermore, some miRNAs were highly enriched (FDR milk whey (3), cells (11) and mammary gland tissue (14) suggesting specific regulatory functions in the various fractions. In conclusion, we have obtained a comprehensive miRNA profile of the different milk fractions using high throughput sequencing. Our comparative analysis showed that miRNAs from milk fat accurately portrayed the miRNome of mammary gland tissue. Functional annotation of the top expressed miRNAs in milk confirmed their critical regulatory roles in mammary gland functions and potentially to milk recipients. PMID:27100870

  16. Toxicological effects of fumonisin B1 alone and in combination with other fusariotoxins on bovine granulosa cells.

    Science.gov (United States)

    Albonico, Marco; Schütz, Luis F; Caloni, Francesca; Cortinovis, Cristina; Spicer, Leon J

    2016-08-01

    There is now overwhelming evidence of global contamination of commodities with Fusarium mycotoxins. Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON), α-zearalenol (α-ZEA) and β-zearalenol (β-ZEA). The aim of this study was to determine if FB1, alone and combined with DON or α-ZEA or β-ZEA, can affect cell proliferation and steroid production of bovine granulosa cells (GC). A species-specific model with bovine granulosa cells (GC) was used to study the potential endocrine disruptor effects of FB1 alone and in co-exposure. In the presence of β-ZEA (30 ng/mL), FB1 at 30 ng/mL showed a stimulatory effect on GC numbers. Insulin-like growth factor-1 (IGF1)-stimulated cell proliferation was decreased after exposure to β-ZEA alone at 5.0 μg/mL and FB1 with α-ZEA and β-ZEA at the same concentration. Regarding steroid production, FB1 at 30 ng/mL and 100 ng/mL amplified the inhibitory effect of β-ZEA (30 ng/mL) on estradiol (E2) production, while FB1 alone increased (P mycotoxin FB1 along with other mycotoxins can affect GC proliferation and steroid production, which ultimately could influence reproductive function in cattle. PMID:27108238

  17. Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

    Directory of Open Access Journals (Sweden)

    Li CongJun

    2006-09-01

    Full Text Available Abstract Background Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences. Results 450 genes significantly regulated by butyrate with a median False Discovery Rate (FDR = 0 % were identified. The majority of these genes were repressed by butyrate and associated with cell cycle control. The expression levels of 30 selected genes identified by the microarray were confirmed using real-time PCR. The results from real-time PCR positively correlated (R = 0.867 with the results from the microarray. Conclusion This study presented the genes related to multiple signal pathways such as cell cycle control and apoptosis. The profound changes in gene expression elucidate the molecular basis for the pleiotropic effects of butyrate on biological processes. These findings enable better recognition of the full range of beneficial roles butyrate may play during cattle energy metabolism, cell growth and proliferation, and possibly in fighting gastrointestinal pathogens.

  18. Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

    Science.gov (United States)

    Lavranos, T C; Mathis, J M; Latham, S E; Kalionis, B; Shay, J W; Rodgers, R J

    1999-08-01

    We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells. PMID:10411512

  19. Differential sensitivities of the prostacyclin and nitric oxide biosynthetic pathways to cytosolic calcium in bovine aortic endothelial cells.

    OpenAIRE

    Parsaee, H.; McEwan, J R; Joseph, S.; MacDermot, J

    1992-01-01

    1. Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; Kact = 0.7 nM) in a concentration-dependent manner when exposed to bradykinin. 2. The bradykinin-dependent release of PGI2 (but not EDRF) was inhibited by 1 microM isoprenaline or 5 microM forskolin, and the inhibitory effect of isoprenaline could be reversed by the beta 2-adrenoceptor antagonist, ICI 118551. In contrast, i...

  20. The effect of yacon (Samallanthus sonchifolius) ethanol extract on cell proliferation and migration of C6 glioma cells stimulated with fetal bovine serum

    OpenAIRE

    Lee, Kang Pa; Choi, Nan Hee; Kim, Jin Teak; Park, In-Sik

    2015-01-01

    BACKGROUND/OBJECTIVES Yacon (Samallanthus sonchifolius), a common edible plant grown throughout the world, is well known for its antidiabetic properties. It is also known to have several other pharmacological properties including anti-inflammatory, anti-oxidant, anti-allergic, and anti-cancer effects. To date, the effect of yacon on gliomas has not been studied. In this study, we investigated the effects of yacon on the migration and proliferation of C6 glioma cells stimulated by fetal bovine...

  1. 2,3,7,8-tetrachlorodibenzo-p-dioxin induced autophagy in a bovine kidney cell line.

    Science.gov (United States)

    Fiorito, Filomena; Ciarcia, Roberto; Granato, Giovanna Elvira; Marfe, Gabriella; Iovane, Valentina; Florio, Salvatore; De Martino, Luisa; Pagnini, Ugo

    2011-12-18

    The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK) cells where no signs of apoptosis were observed, but herein, analysis of MDBK cell morphology, in a large number of exposed cells, revealed some alterations, as expanded cytoplasm, an increase of intercellular spaces and many pyknotic nuclei. Hence, the aim of the current study was to elucidate the influences of dioxin on cell proliferation and cell death. We found that dioxin increased proliferation, as well as, activated cell death with autophagy, as we detected by increased amount of LC3-II, an autophagosome marker. Furthermore, formation of acidic vesicular organelles was observed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. These results were accompanied by down-regulation of telomerase activity, bTERT and c-Myc. Key tumor-suppressor protein p53 and expression of cell cycle inhibitor p21Waf1/Cip1 were activated after TCDD exposure. These changes occurred with activation of ATM phosphorylation in the presence of a decrease in Mdm2 protein levels. Taken together, these results support the idea that TCDD in MDBK cells, may exert its action, in part, by enhancing cell proliferation, but also by modulating the incidence of induced cell death with autophagy.

  2. Highly Efficient In Vitro Production of Bovine Blastocyst in Cell-Free Sequential Synthetic Oviductal Fluid vs. TCM199 Vero Cell Co-Culture System

    Directory of Open Access Journals (Sweden)

    Sayyed Morteza Hosseini

    2008-01-01

    Full Text Available Background: The aim of this study was to establish a cell-free sequential culture system that cansupport high levels of in vitro embryo development and blastocyst formation from bovine zygotes.To this end, this investigation was carried out to evaluate the effects of glucose, serum and EDTAon bovine zygote in vitro development.Materials and Methods: Bovine presumptive zygotes were derived from oocytes matured, andfertilized in vitro and cultured in synthetic oviductal fluid sequential medium in a two-steps manner;SOF 1 for the first 3 days and SOF 2 for the second 5-6 days of in vitro embryo development. Inorder to evaluate the effect of different modifications of the basic medium on embryo development,glucose was added to the second phase (SOF A, serum was added to the first phase (SOF C andEDTA alone (SOF D or in combination with serum (SOF E was added into the first phase of invitro embryo culture. The results of each composition were compared with each other and with theresults of embryo development in TCM199 vero cell co-culture system.Results: Glucose addition to the second phase of embryo culture, improved the developmentalcompetency; however, the differences were not significant. Serum addition to the first phase ofembryo culture, significantly improved the developmental competency of embryos beyond thecleavage stage, compared to all the treatment and TCM199 co-culture groups. EDTA supplementationof culture medium, either alone or in combination with serum, significantly inhibits the embryodevelopment beyond the morula stage.Conclusion: The results indicated that culture of bovine presumptive zygotes in two steps cell-freeculture system, can support embryo development, and addition of serum throughout the culture andglucose to the second step significantly increased overall developmental competency compared toTCM199 co-culture system.

  3. Identification of genes differentially expressed in myogenin knock-down bovine muscle satellite cells during differentiation through RNA sequencing analysis.

    Directory of Open Access Journals (Sweden)

    Eun Ju Lee

    Full Text Available BACKGROUND: The expression of myogenic regulatory factors (MRFs consisting of MyoD, Myf5, myogenin (MyoG and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. Although it is well known that the function of MyoG cannot be compensated for other MRFs, the molecular mechanism by which MyoG controls muscle cell differentiation is still unclear. Therefore, in this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knock-down (MyoGkd in primary bovine muscle satellite cells (MSCs. RESULTS: About 61-64% of the reads of over 42 million total reads were mapped to more than 13,000 genes in the reference bovine genome. RNA-Seq analysis identified 8,469 unique genes that were differentially expressed in MyoGkd. Among these genes, 230 were up-regulated and 224 were down-regulated by at least four-fold. DAVID Functional Annotation Cluster (FAC and pathway analysis of all up- and down-regulated genes identified overrepresentation for cell cycle and division, DNA replication, mitosis, organelle lumen, nucleoplasm and cytosol, phosphate metabolic process, phosphoprotein phosphatase activity, cytoskeleton and cell morphogenesis, signifying the functional implication of these processes and pathways during skeletal muscle development. The RNA-Seq data was validated by real time RT-PCR analysis for eight out of ten genes as well as five marker genes investigated. CONCLUSIONS: This study is the first RNA-Seq based gene expression analysis of MyoGkd undertaken in primary bovine MSCs. Computational analysis of the differentially expressed genes has identified the significance of genes such as SAP30-like (SAP30L, Protein lyl-1 (LYL1, various matrix metalloproteinases, and several glycogenes in myogenesis. The results of the present study widen our knowledge of the molecular basis of skeletal muscle

  4. [Comparison of methods of detection of bovine adenovirus serotype 3 in infected culture of calf kidney cells (MDBK)].

    Science.gov (United States)

    Kaliuzhnaia, A N; Trifonov, V D; Zil'berman, M I; Zalmanzon, E S; Kaledin, A S

    1988-10-01

    The comparative study of the dynamics of the main antigen (hexon) and viral DNA of the bovine adenovirus type 3 accumulation in the established cell line MDBK under the conditions of single- or multistep cycle of infection has been undertaken. The quantitative immunoelectrophoresis and immunoenzyme assay detected the viral antigens on the late stages of infection in the period of cellular monolayer degradation. The immunofluorescence reaction and histochemical immunoenzyme method detected the antigen in the infected cells concurrently with the primary expression of the viral cytopathic effect. The reaction of the spot molecular hybridization with the [32P]-DNA probes detected the viral DNA considerably earlier than the antigen was detected by the immunological methods, before the appearance of degenerative changes in the infected cells. Preference of the immunoenzyme assay and DNA-probes in diagnosis of the virus are discussed.

  5. Giardia duodenalis stimulates partial maturation of bovine dendritic cells associated with altered cytokine secretion and induction of T-cell proliferation.

    Science.gov (United States)

    Grit, G H; Devriendt, B; Van Coppernolle, S; Geurden, T; Hope, J; Vercruysse, J; Cox, E; Geldhof, P; Claerebout, E

    2014-04-01

    Giardia duodenalis is an important intestinal parasite in animals and humans. The role of dendritic cells (DC) in the initiation of the immune response against G. duodenalis is poorly documented. The aim of this study was to test the hypothesis that G. duodenalis interferes with bovine DC function. Therefore, the effect of trophozoites and excretion/secretion products on bovine monocyte-derived dendritic cells (MoDC) was investigated. We assessed MoDC maturation and cytokine production of G. duodenalis-stimulated MoDC and the ability of these MoDC to take up antigen and induce lymphocyte proliferation. Little or no upregulation of maturation markers CD40 and CD80 was measured, but MHCII expression was increased after stimulation with low parasite concentrations. A dose-dependent decrease in ovalbumin uptake was observed in G. duodenalis-stimulated MoDC. In addition, stimulated MoDC induced proliferation of CD3(-) , γδ-T-cells and TCRαβ(+) CD4(+) and CD8(+) T-cells. Increased transcription of TGF-β was shown in CD4(+) T cells, and increased TNF-α, TGF-β, IL-10 and IL-4 were seen in γδ-T-cells. We found no evidence that G. duodenalis has a regulatory or inhibitory effect on bovine MoDC. MoDC stimulated with G. duodenalis are functionally active and able to induce proliferation of T cells that produce both pro- and anti-inflammatory cytokines.

  6. Differentiation-specific action of orphan nuclear receptor NR5A1 (SF-1): transcriptional regulation in luteinizing bovine theca cells

    OpenAIRE

    Walther, Norbert; Jansen, Martina; Akbary, Wasima; Ivell, Richard

    2006-01-01

    Background The orphan nuclear receptor NR5A1 (steroidogenic factor-1, SF-1) is a master regulator of tissue-specific gene expression in reproductive and steroidogenic tissues. Two activating functions, AF-1 and AF-2, have been described to function in a cooperative manner to recruit transcriptional coactivators to the promoter regions of NR5A1-controlled genes. Methods The role of the NR5A1 activating functions AF-1 and AF-2 was studied in primary bovine theca cells. Bovine theca cells were i...

  7. Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200® implanted heifers

    Institute of Scientific and Technical Information of China (English)

    Andrea DStapp; Craig AGifford; Dennis MHallford; Jennifer AHernandez Gifford

    2014-01-01

    Background:Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments:non-implanted (n=5), Revalor 200 for 28 d (n=5), or Revalor 200 for 84 d (n=6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n=16) or 100 ng/mL follicle stimulating hormone (n=16) for 24 h. Results:Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P=0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P<0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P=0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P<0.10) or 84 d (P<0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P=0.57) and aromatase (P=0.23) were demonstrated in implanted or non-implanted heifers. Conclusions:These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle

  8. Proteomic Analysis of Bovine Nucleolus

    Institute of Scientific and Technical Information of China (English)

    Amrutlal K.Patel; Doug Olson; Suresh K. Tikoo

    2010-01-01

    Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eu-karyotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells,we analyzed the proteomie composition of the bovine nueleoli. The nucleoli were isolated from Madin Darby bo-vine kidney cells and subjected to proteomie analysis by LC-MS/MS after fractionation by SDS-PAGE and strongcation exchange chromatography. Analysis of the data using the Mascot database search and the GPM databasesearch identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in theproteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggestedthat the bovine nueleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional,translational and post-translational regulation, transport, and structural organization.

  9. Immunohistochemical visualization of insulin receptors in formalin-fixed bovine ovaries post mortem and in granulosa cells collected in vivo.

    Science.gov (United States)

    Bossaert, P; De Cock, H; Leroy, J L M R; De Campeneere, S; Bols, P E J; Filliers, M; Opsomer, G

    2010-06-01

    Insulin is crucial for granulosa cell (GC) function, follicle growth and ovulation in cows; low insulin levels increase the risk for anoestrus. Apart from insulin concentration, alterations in the insulin receptor (IR) density on GC may affect follicular growth and steroidogenesis. Data about the IR protein distribution in the bovine follicle are scarce. Therefore, we aimed to develop a quantifiable staining method for IR protein on histological sections of bovine follicles in different developmental stages, and to apply this technique on GC obtained in living cows. In a first experiment, bovine ovaries were collected post mortem, formalin fixed, routinely processed, and stained with monoclonal murine IR-antibodies, peroxidase-labeled goat anti-mouse antibodies, and substrate chromogen. Based on their diameter, follicles were morphologically classified as small antral (SAF; n = 141), dominant (DF; n=28) or subordinate (SF; n=8); DF and SF were further classified as healthy or atretic based on the ratio of estrogen and progesterone concentrations in their follicular fluid. Using specialized software, the proportion of pixels displaying a positive staining signal was computed as a measure for IR density in three selected follicular regions: GC, theca (T) and stroma (STR). Results were analyzed in an ANOVA model with follicle type, region and health status as fixed factors. In SAF, DF, and SF, IR density was notably higher in GC than T or STR; the latter two displayed very low or no IR presence. The IR density in SAF was stronger than in DF and tended to be stronger than in SF. Staining intensity was not altered in atretic compared to healthy follicles. In corpus luteum, cumulus-oocyte complexes and pre-antral follicles, no IR could be detected. In a second experiment, GC samples were collected from 20 live cows on 30 and 70 d post partum by transvaginal follicular fluid aspiration, projected on glass slides, and stained using the protocol described above. Most

  10. Effect of the Ketone Body Beta-Hydroxybutyrate on the Innate Defense Capability of Primary Bovine Mammary Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Maria Hillreiner

    Full Text Available Negative energy balance and ketosis are thought to cause impaired immune function and to increase the risk of clinical mastitis in dairy cows. The present in vitro study aimed to investigate the effect of elevated levels of the predominant ketone body β-hydroxybutyrate on the innate defense capability of primary bovine mammary epithelial cells (pbMEC challenged with the mastitis pathogen Escherichia coli (E. coli. Therefore, pbMEC of healthy dairy cows in mid- lactation were isolated from milk and challenged in culture with 3 mM BHBA and E. coli. pbMEC stimulated with E. coli for 6 h or 30 h showed an up-regulation of several innate immune genes, whereas co-stimulation of pbMEC with 3 mM BHBA and E. coli resulted in the down-regulation of CCL2, SAA3, LF and C3 gene expression compared to the challenge with solely the bacterial stimulus. These results indicated that increased BHBA concentrations may be partially responsible for the higher mastitis susceptibility of dairy cows in early lactation. Elevated levels of BHBA in blood and milk during negative energy balance and ketosis are likely to impair innate immune function in the bovine mammary gland by attenuating the expression of a broad range of innate immune genes.

  11. Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

    Directory of Open Access Journals (Sweden)

    Edda de Rizzo

    1984-04-01

    Full Text Available Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE. As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80 than the latter (1:80 to 1:160. Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

  12. Expression and regulation of regulator of G-protein signaling protein-2 (RGS2) in equine and bovine follicles prior to ovulation: molecular characterization of RGS2 transactivation in bovine granulosa cells.

    Science.gov (United States)

    Sayasith, Khampoun; Sirois, Jean; Lussier, Jacques G

    2014-12-01

    The luteinizing hormone preovulatory surge stimulates several signal pathways essential for ovulation, and the regulator of G-protein signaling protein-2 (RGS2) is thought to be involved in this process. The objectives of this study were to characterize the regulation of RGS2 transcripts in equine and bovine follicles prior to ovulation and to determine its transcriptional control in bovine granulosa cells. To assess the regulation of equine RGS2 prior to ovulation, RT-PCR was performed using total RNA extracted from equine follicles collected at various times after human chorionic gonadotropin (hCG) injection. Results showed that RGS2 mRNA levels were very low at 0 h but markedly increased 12-39 h post-hCG (P < 0.05). In the bovine species, results revealed that RGS2 mRNA levels were low in small and dominant follicles and in ovulatory follicles obtained at 0 h, but markedly increased in ovulatory follicles 6-24 h post-hCG (P < 0.05). To study the molecular control of RGS2 expression, primary cultures of bovine granulosa cells were used. Stimulation with forskolin induced an up-regulation of RGS2 mRNA in vitro. Studies using 5'-deletion mutants identified a minimal region containing full-length basal and forskolin-inducible RGS2 promoter activities. Site-directed mutagenesis indicated that these activities were dependent on CRE and ETS1 cis-elements. Electrophoretic mobility shift assays confirmed the involvement of these elements and revealed their interactions with CREB1 and ETS1 proteins. Chromatin immunoprecipitation assays confirmed endogenous interactions of these proteins with the RGS2 promoter in granulosa cells. Forskolin-inducible RGS2 promoter activity and mRNA expression were markedly decreased by PKA and ERK1/2 inhibitors, and treatment with an antagonist of PGR (RU486) and inhibitors of PTGS2 (NS398) and EGFR (PD153035) blocked the forskolin-dependent RGS2 transcript expression, suggesting the importance of RGS2 in ovulation. Collectively, this study

  13. 奶牛乳腺上皮细胞系的培养与鉴定%Culture and Identification of the Bovine Mammary Epithelial Cell Line

    Institute of Scientific and Technical Information of China (English)

    詹康; 贡笑笑; 左晓昕; 陈银银; 占今舜; 赵国琦

    2015-01-01

    本试验旨在培养功能性奶牛乳腺上皮细胞系,为奶牛泌乳调控和奶牛乳房炎发病机制研究提供功能性的细胞模型。采用组织块细胞培养法来分离纯化并鉴定奶牛乳腺上皮细胞;采用有限稀释法单克隆奶牛乳腺上皮细胞;采用噻唑蓝( MTT)法来分析奶牛乳腺上皮细胞的生长曲线是否为正常的“S”形;观察细胞角蛋白18免疫荧光来证明所培养的细胞为上皮型;选择培养至10和20代的奶牛乳腺上皮细胞进行染色体核型分析。结果表明:1)利用组织块细胞分离法能够成功获得奶牛乳腺上皮细胞并传至20代。2)培养5~6 d成纤维细胞迅速增殖且周围分裂出少量的上皮细胞。培养8d奶牛乳腺上皮细胞迅速增殖,形成岛屿状集落,呈单层“鹅卵石”和“铺路石”形态生长。3)奶牛乳腺上皮细胞角蛋白18鉴定为阳性。4)培养至10和20代的奶牛乳腺上皮细胞染色体数为60条,具有正常的细胞二倍体核型。综上所述,采用组织块培养细胞能够获得具有稳定性、功能性的奶牛乳腺上皮细胞,但不是永生化细胞。%To establish a functional model of bovine mammary epithelial cell line for the study of lactation reg-ulation, pathogenesis of mastitis, this research applied tissue culture to isolate and culture bovine mammary epi-thelial cells; the limiting dilution method to purify bovine mammary epithelial cells; methyl thiazolyl tetrazoli-um ( MTT) method was used to identify whether the growth curve of bovine mammary epithelial cells was‘S’ type or not;cytokeratin 18 immunofluorescence was observed to prove that the cells were epithelial type;kary-otype analysis was carried out on bovine mammary epithelial cells in passages 10 and 20. The results showed as follows:1) bovine mammary epithelial cells could be successfully cultured and passaged 20 generations by tis-sue culture method. 2) At 5 to 6 days of cultivation, the

  14. DEXAMETHASONE DIFFERENTIALLY DOWN-REGULATES L-SELECTIN (CD62L) EXPRESSION BY BOVINE LYMPHOCYTE SUBSETS IN VIVO AND DEPLETES THE INTESTINAL MUCOSA OF INTRAEPITHELIAL GAMMA DELTA CELLS

    Science.gov (United States)

    A hallmark of immune modulation by dexamethasone (DEX) is the suppression of lymphocyte proliferation. DEX also alters the expression of adhesion molecules on bovine lymphocytes. Although intraepithelial lymphocytes (IEL) mainly are non-proliferating cells, we hypothesized that DEX treatment of ca...

  15. Bovine viral diarrhea virus type 2 in vivo infection modulates TLR4 responsiveness in differentiated Myeloid cells which is associated with decreased MyD88 expression

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV) causes clinical signs in cattle ranging from mild to severe acute infection which can lead to increased susceptibility to secondary bacteria. In this study we examined the effects of BVDV genotype 2 (BVDV2) infection on the ability of myeloid lineage cells derived...

  16. Comparison of flow cytometry and virus isolation in cell culture for identification of cattle persistently infected with bovine viral diarrhea virus.

    OpenAIRE

    Qvist, P.; Houe, H.; Aasted, B.; Meyling, A

    1991-01-01

    Detection of bovine viral diarrhea virus in 143 blood samples by virus isolation in cell culture and flow cytometry was performed. The material included 37 samples later shown to originate from persistently infected cattle. Thirty-three samples were positive by virus isolation, and all 37 samples were positive by the flow cytometric assay.

  17. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    Science.gov (United States)

    Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina

    2016-01-01

    Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients. PMID:27191003

  18. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    Directory of Open Access Journals (Sweden)

    Erika Evangelina Coronado-Cerda

    2016-01-01

    Full Text Available Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE or IMMUNEPOTENT CRP® (ICRP is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM, cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients.

  19. The anti-epileptic drug valproic acid (VPA inhibits steroidogenesis in bovine theca and granulosa cells in vitro.

    Directory of Open Access Journals (Sweden)

    Claire Glister

    Full Text Available Valproic acid (VPA is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC and granulosa (GC cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P99% decrease; P<0.0001 with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05. VPA only reduced TC progesterone secretion induced by the highest (luteinizing LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001 by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001. The potent histone deacetylase (HDAC inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

  20. Optimization and characterization of an in vitro bovine mammary cell culture system to study regulation of milk protein synthesis and mammary differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Talhouk, R.S.

    1988-01-01

    A long term bovine mammary cell culture system that maintains normal mammary cell function was established and optimized to study milk protein synthesis and secretion and mammary differentiation. This culture system used bovine mammary acini isolated from developing or lactating mammary gland by enzymatic dissociation, and cryopreserved until thawed and plated for growth in vitro for these studies. Cells in M199 with lactogenic hormones {plus minus} fetal calf serum (FCS) were cultured on plastic, 100ul and 500ul type I collagen, and Matrigel, or embedded within type I collagen. Cell morphology, cell number, and total TCA-precipitable {sup 35}S-labelled proteins were monitored. Milk protein ({alpha}{sub s,1}-casein, lactoferrin (LF), {alpha}-lactalbumin, and {beta}-lactoglobulin) secretion and intracellular levels were determined by an ELISA assay.

  1. The Inhibition Effect of Cell DNA Oxidative Damage and LDL Oxidation by Bovine Colostrums

    Directory of Open Access Journals (Sweden)

    Chih-Wei Chen

    2016-10-01

    Full Text Available In the present study, we investigated the effect of bovine colostrums on inhibition of DNA oxidative damage and low density lipoprotein (LDL oxidation in vitro. Results showed that whey and skimmed milk exhibited not only higher inhibitory activities of oxidative damage of deoxyribose but also an inhibitory effect on the breakdown of supercoiled DNA into open circular DNA and linear DNA. The quantities of 8-OH-2′-dG formed under whey, caseins and skimmed milk treatment were 0.24, 0.24 and 1.24 μg/mL, respectively. The quantity of malondialdehyde formed through LDL oxidation induced by copprous ion was significantly decreased as colostrums protein solutions were added, in which whey and caseins led to a more significant decrease than skimmed milk. The formation of conjugated dienes could be inhibited by treatment with colostrums protein solutions. Whey exhibited the longest lag time of conjugated dienes formation among the colostrums proteins. The lag time of the whey was 2.33 times that of the control. From the results of foregoing, the bovine colostrums protein has potential value in the inhibition of DNA oxidation damage and LDL oxidation.

  2. BOVINE VIRAL DIARRHEA VIRUSES

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV) is an umbrella term for two species of viruses, BVDV1 and BVDV2, within the Pestivirus genus of the Flavivirus family. BVDV viruses are further subclassified as cytopathic and noncytopathic based on their activity in cultured epithelial cells. Noncytopathic BVDV p...

  3. Cooperation of hTERT, SV40 T Antigen and Oncogenic Ras in Tumorigenesis: A Cell Transplantation Model Using Bovine Adrenocortical Cells

    Directory of Open Access Journals (Sweden)

    Michael Thomas

    2002-01-01

    Full Text Available Expression of TERT, the reverse transcriptase component of telomerase, is necessary to convert normal human cells to cancer cells. Despite this, “telomerization” by hTERT does not appear to alter the normal properties of cells. In a cell transplantation model in which bovine adrenocortical cells form vascularized tissue structures beneath the kidney capsule in scid mice, telomerization does not perturb the functional tissue-forming capacity of the cells. This cell transplantation model was used to study the cooperation of hTERT with SV40 T antigen (SV40 TAg and oncogenic Ras in tumorigenesis. Only cells expressing all three genes were tumorigenic; this required large T, but not small t, antigen. These cells produced a continuously expanding tissue mass; they were invasive with respect to adjacent organs and eventually destroyed the kidney. Cells expressing only hTERT or only Ras produced minimally altered tissues. In contrast, SV40 TAg alone produced noninvasive nodules beneath the kidney capsule that had high proliferation rates balanced by high rates of apoptosis. The use of cell transplantation techniques in a cell type that is able to form tissue structures with or without full neoplastic conversion allows the phenotypes produced by individual cooperating oncogenes to be observed.

  4. IGF-binding proteins mediate TGF-beta 1-induced apoptosis in bovine mammary epithelial BME-UV1 cells.

    Science.gov (United States)

    Gajewska, Małgorzata; Motyl, Tomasz

    2004-10-01

    TGF-beta 1 is an antiproliferative and apoptogenic factor for mammary epithelial cells (MEC) acting in an auto/paracrine manner and thus considered an important local regulator of mammary tissue involution. However, the apoptogenic signaling pathway induced by this cytokine in bovine MEC remains obscure. The present study was focused on identification of molecules involved in apoptogenic signaling of transforming growth factor-beta 1 (TGF-beta 1) in the model of bovine mammary epithelial cell line (BME-UV1). Laser scanning cytometry (LSC), Western blot and electrophoretic mobility shift assay (EMSA) were used for analysis of expression and activity of TGF-beta 1-related signaling molecules. The earliest response occurring within 1-2 h after TGF-beta 1 administration was an induction and activation of R-Smads (Smad2 and Smad3) and Co-Smad (Smad4). An evident formation of Smad-DNA complexes began from 2nd hour after MEC exposure to TGF-beta 1. Similarly to Smads, proteins of AP1 complex: phosphorylated c-Jun and JunD appeared to be early reactive molecules; however, an increase in their expression was detected only in cytosolic fraction. In the next step, an increase of IGF binding protein-3 (IGFBP-3) and IGFBP-4 expression was observed from 6th hour followed by a decrease in the activity of protein kinase B (PKB/Akt), which occurred after 24 h of MEC exposure to TGF-beta 1. The decrease in PKB/Akt activity coincided in time with the decline of phosphorylated Bad expression (inactive form). Present study supported additional evidence that stimulation of insulin-like growth factor I (IGF-I) was associated with complete abrogation of TGF-beta 1-induced activation of Bad and Bax and in the consequence protection against apoptosis. In conclusion, apoptotic effect of TGF-beta 1 in bovine MEC is mediated by IGFBPs and occurs through IGF-I sequestration, resulting in inhibition of PKB/Akt-dependent survival pathway. PMID:15556067

  5. The effect of beta-turn structure on the permeation of peptides across monolayers of bovine brain microvessel endothelial cells

    DEFF Research Database (Denmark)

    Sorensen, M; Steenberg, B; Knipp, G T;

    1997-01-01

    PURPOSE: To investigate the effects of the beta-turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). METHODS: The effective...... permeability coefficients (Peff) of the model peptides were determined across BBMEC monolayers. The dimensions of the aqueous pores in the tight junctions (TJs) of the BBMEC monolayers were determined using a series of hydrophilic permeants. This value and the molecular radius of each peptide were used...... to calculate the theoretical paracellular (PP*) and transcellular (PT*) permeability coefficients for each peptide. RESULTS: A comparison of the theoretical PP* values with the observed Peff values was made for a series of model peptides. For the most hydrophobic peptides (Ac-PheProXaaIle-NH2 and Ac...

  6. α-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. I. Purification from conditioned medium

    International Nuclear Information System (INIS)

    A 6-kDa α-transforming growth factor (TGF) was purified 100,000-fold to homogeneity from the culture fluid conditioned by normal bovine anterior pituitary-derived cells. Initial purification of the acid-soluble TGF from concentrated conditioned medium was achieved by Bio-Gel P-60 gel filtration (apparent molecular mass of 9 kDa). After the Bio-Gel step, three different steps of reverse-phase fast-protein liquid chromatography on the same Pharmacia C18 column, using linear acetonitrile gradients, gave complete purification. The ion-pairing agents used in the three consecutive steps were: 0.1% trifluoroacetic acid, 0.13% heptafluorobutyric acid, and again, 0.1% trifluoroacetic acid at a shallower gradient. Homogeneity was confirmed by reverse-phase high performance liquid chromatography, and by polyacrylamide gel electrophoresis, where TGF visualization was facilitated by autoradiography of 125I-TGF. The 125I-TGF bound to epidermal growth factor (EGF) receptors and after elution ran identically to the starting material. The molecular mass of TGF is 6 kDa by polyacrylamide gel electrophoresis and 6.6 kDa by amino acid analysis. The amino acid composition of bovine TGF is similar to that of rat or human αTGF and distinct from epidermal growth factor. Colony-stimulating activity was lost after purification, but the TGF retained its ability to stimulate thymidine uptake by quiescent cells. This mitogenic activity could be blocked completely by anti-EGF-receptor monoclonal antibodies, indicating that the activity was mediated through the EGF-receptor

  7. Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle

    Directory of Open Access Journals (Sweden)

    Marlene I. Vargas

    2014-10-01

    Full Text Available Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A were intravenously inoculated with 6.6 X10(7 red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1, for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP was measured in bovine umbilical vein endothelial cells (BUVECs. The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.

  8. Regulation of Innate Immune Responses by Bovine Herpesvirus 1 and Infected Cell Protein 0 (bICP0

    Directory of Open Access Journals (Sweden)

    Clinton Jones

    2009-09-01

    Full Text Available Bovine herpesvirus 1 (BoHV-1 infected cell protein 0 (bICP0 is an important transcriptional regulatory protein that stimulates productive infection. In transient transfection assays, bICP0 also inhibits interferon dependent transcription. bICP0 can induce degradation of interferon stimulatory factor 3 (IRF3, a cellular transcription factor that is crucial for activating beta interferon (IFN-β promoter activity. Recent studies also concluded that interactions between bICP0 and IRF7 inhibit trans-activation of IFN-β promoter activity. The C3HC4 zinc RING (really important new gene finger located near the amino terminus of bICP0 is important for all known functions of bICP0. A recombinant virus that contains a single amino acid change in a well conserved cysteine residue of the C3HC4 zinc RING finger of bICP0 grows poorly in cultured cells, and does not reactivate from latency in cattle confirming that the C3HC4 zinc RING finger is crucial for viral growth and pathogenesis. A bICP0 deletion mutant does not induce plaques in permissive cells, but induces autophagy in a cell type dependent manner. In summary, the ability of bICP0 to stimulate productive infection, and repress IFN dependent transcription plays a crucial role in the BoHV-1 infection cycle.

  9. Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments

    Institute of Scientific and Technical Information of China (English)

    San-Hua Leng; Fu-Er Lu

    2005-01-01

    AIM: To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study.METHODS: The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine oells with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels(KATP), voltage-dependent potassium channels (KV), and voltage-dependent calcium channels (KCA) in β-cells were identified by patch clamp technique.RESULTS: After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly (MTT value from 0.024±0.003 to0.028±0.003, insulin secretion from 2.6±0.6to 3.1±0.8 mIU/L). Then MTT value and insulin secretion increased quickly from d 5 to d 10 (MTT value from 0.028±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8to 18.3±2.6 mIU/L), then reached high plateau (MTT value >0.052±0.008, insulin secretion >18.3±2.6 mIU/L).In contrast, for the isolated adult pancreatic islet cells,both insulin release and MTT value were stable in the first 4 d (MTT value from 0.029±0.01 to 0.031±0.011,insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L), but afterwards they reduced gradually (MTT value <0.031±0.011, insulin secretion <8.2±1.5 mIU/L), and the pancrearic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP KV, and KCA.CONCLUSION: Islet

  10. Islet-activating protein inhibits leukotriene D4- and leukotriene C4- but not bradykinin- or calcium ionophore-induced prostacyclin synthesis in bovine endothelial cells.

    OpenAIRE

    Clark, M. A.; Conway, T.M.; Bennett, C F; Crooke, S T; Stadel, J M

    1986-01-01

    Incubation of the bovine endothelial cell line, CPAE, with leukotriene D4, leukotriene C4, bradykinin, or the calcium ionophore A23187 results in the release of arachidonic acid metabolites including 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. Pretreatment of these cells with the pertussis toxin islet-activating protein (IAP) results in a dose-dependent inhibition of the release of arachidonic acid metabolites and prostacyclin in response to leukotriene D4 and leukot...

  11. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Science.gov (United States)

    Chou, Ming-Li; Burnouf, Thierry; Wang, Tsung-Jen

    2014-01-01

    Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory

  12. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Directory of Open Access Journals (Sweden)

    Ming-Li Chou

    Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices

  13. Assessment of the nucleotide sequence variability in the bovine T-cell receptor alpha delta joining gene region.

    Science.gov (United States)

    Fries, R; Ewald, D; Thaller, G; Buitkamp, J

    2001-05-01

    The sequence of 2,193 nucleotides from the bovine T-cell receptor alpha/delta joining gene region (TCRADJ) was determined and compared with the corresponding human and murine sequences. The identity was 75.3% for the comparison of the Bos taurus vs. the Homo sapiens sequence and 63.8% for the Bos taurus vs. the Mus musculus sequence. This comparison permitted the identification of the putatively functional elements within the bovine sequence. Direct sequencing of 2,110 nucleotides in nine animals revealed 12 variable sites. Estimates, based on direct sequencing in three Holstein Friesian animals, for the two measures of sequence variability, nucleotide polymorphism (u) and nucleotide diversity (p), were 0.00050 (60.00036) and 0.00077 (60.00056), respectively. The test statistic, Tajima's D, for the comparison of the two measures indicates that the difference between u and p is close to significance (P Braunvieh, 115 Fleckvieh, 47 Holstein Friesian, 50 Simmental and 43 Pinzgauer) using the oligonucleotide ligation assay (OLA) in combination with the enzyme linked immunoabsorbant assay (ELISA). Nine unambiguous haplotypes could be derived based on animals with a maximum of one heterozygous site. Four to seven haplotypes were present in the different breeds. When taking into account the frequencies of the haplotypes in the different breeds, especially in Anatolian Black, an ancestral cattle population, we could establish the likely phylogenetic relationships of the haplotypes. Such haplotype trees are the basis for cladistic candidate gene analysis. Our study demonstrates that the systematic search of single nucleotide polymorphisms (SNPs) is useful for analysing all aspects of variability of a given genomic region.

  14. Different screening tests and milk somatic cell count for the prevalence of subclinical bovine mastitis in Bangladesh.

    Science.gov (United States)

    Hoque, Md Nazmul; Das, Ziban Chandra; Talukder, Anup Kumar; Alam, Mohammad Shah; Rahman, Abu Nasar Md Aminoor

    2015-01-01

    Identification of cows with subclinical mastitis (SCM) is an important tool for sustainable dairying and implementing effective mastitis control strategies. A total of 892 quarters milk samples from 228 lactating cows were screened by California mastitis test (CMT), White side test (WST), Surf field mastitis test (SFMT), and somatic cell count (SCC) to study the prevalence of bovine SCM in some selected areas of Bangladesh. Out of 228 cows, 148 (64.9%), 138 (60.5%), 132 (57.9%), and 164 (71.9%) were found positive for SCM by CMT, WST, SFMT, and SCC, respectively. The prevalence of bovine SCM was diagnosed 45.7, 40.2, 36.6, and 29.6% in Chittagong, Sirajgonj, Mymensingh, and Gazipur districts, respectively, based on a combination of all tests. The overall quarter-wise prevalence of SCM was 45.7, 43.5, 41.2, and 55.0% for CMT, WST, SFMT, and SCC. Single quarters and left front quarters were more prone to SCM (P CMT, WST, SFMT, and SCC was 65.8, 57.9, 51.0, and 82.5%; specificity 76.2, 72.4, 69.5, and 89.4%; percentage accuracy 70.0, 64.8, 59.9, and 85.2%; positive predictive value 75.2, 69.8, 64.9, and 92.7%, respectively. The categories of CMT reactions were strongly correlated with SCC (P tests (SCC>CMT>WST>SFMT). Thus, CMT was concluded to be the most accurate (r = 0.782) field diagnostic test after laboratory test like SCC (r = 0.924). However, the use of any single test may not be reliable in diagnosing SCM, while the result of CMT supported by SCC might be used effectively to pinpoint diagnosis of SCM in dairy animals than alone. PMID:25326717

  15. Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization.

    Science.gov (United States)

    Díaz-Murillo, Violeta; Medina-Estrada, Ivan; López-Meza, Joel E; Ochoa-Zarzosa, Alejandra

    2016-04-01

    β-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal β-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant β-defensins remain unknown. In addition, the regulation of the immune system by mammalian β-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized β-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 μg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1β (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of β-defensins from plants as immunomodulators of the mammalian

  16. Supramolecular assembly of biological molecules purified from bovine nerve cells: from microtubule bundles and necklaces to neurofilament networks

    International Nuclear Information System (INIS)

    With the completion of the human genome project, the biosciences community is beginning the daunting task of understanding the structures and functions of a large number of interacting biological macromolecules. Examples include the interacting molecules involved in the process of DNA condensation during the cell cycle, and in the formation of bundles and networks of filamentous actin proteins in cell attachment, motility and cytokinesis. In this proceedings paper we present examples of supramolecular assembly based on proteins derived from the vertebrate nerve cell cytoskeleton. The axonal cytoskeleton in vertebrate neurons provides a rich example of bundles and networks of neurofilaments, microtubules (MTs) and filamentous actin, where the nature of the interactions, structures, and structure-function correlations remains poorly understood. We describe synchrotron x-ray diffraction, electron microscopy, and optical imaging data, in reconstituted protein systems purified from bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories of polyelectrolyte bundling, including three-dimensional MT bundles and two-dimensional MT necklaces

  17. Detection of a Novel Bovine Lymphotropic Herpesvirus

    OpenAIRE

    Rovnak, Joel; Quackenbush, Sandra L.; Reyes, Richard A.; Baines, Joel D.; Parrish, Colin R.; Casey, James W.

    1998-01-01

    Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine h...

  18. VISFATIN (NAMPT) Improves In Vitro IGF1-Induced Steroidogenesis and IGF1 Receptor Signaling Through SIRT1 in Bovine Granulosa Cells.

    Science.gov (United States)

    Reverchon, Maxime; Rame, Christelle; Bunel, Audrey; Chen, Wenyong; Froment, Pascal; Dupont, Joëlle

    2016-03-01

    VISFATIN is a novel adipokine, also known as a nicotinamide phosphorybosyltransferase (NAMPT), that is able to modulate different processes, including lipid and glucose metabolism, oxidative stress, inflammation, and insulin resistance. Recent data suggest that it also plays a role in reproductive function in rats, humans, and chickens. Here we identified VISFATIN in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and proliferation and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found VISFATIN in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, we showed that IGF1 (10(-8) M) and VISFATIN (10 and 100 ng/ml) but not FSH (10(-8) M) increased mRNA expression levels of NAMPT after 48 h of stimulation. Moreover, we observed that human recombinant VISFATIN (hVisf, 10 ng/ml, 48 h) increased the release of progesterone and estradiol secretion, and this was associated with an increase in the protein level of STAR, the HSD3B activity, and the phosphorylation levels of IGF1R and MAPK ERK1/2 in the presence or absence of IGF1 (10(-8) M). All these effects were abolished when NAMPT was knocked down and when the sirtuin pharmacological inhibitors CHIC-35 (60 nM) and EX-527 (0.5 μM) were preincubated in bovine granulosa cells. Thus, in cultured bovine granulosa cells, VISFATIN improves basal and IGF1-induced steroidogenesis and IGF1 receptor signaling through SIRT1. PMID:26792944

  19. α-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. II. Identification using a sequence-specific monoclonal antibody

    International Nuclear Information System (INIS)

    Untransformed bovine anterior pituitary cells cultured in serum-free defined medium secrete an epidermal growth factor (EGF)-like peptide with an amino acid composition similar to rat or human α-transforming growth factor (αTGF). To further characterize the bovine pituitary αTGF, it was compared to a human αTGF partially purified from the conditioned medium of a human melanoma cell line. An anti-αTGF monoclonal antibody, MF9, was produced from hybridomas derived from mice immunized with a 17-residue synthetic peptide corresponding to the carboxyl-terminal sequence of rat αTGF. The hybridoma supernatants were initially screened for the ability to immunoprecipitate 125I-peptide and then tested for recognition of human αTGF. Only 2 of 36 antipeptide antibodies recognized the native αTGF. The binding of 125I-peptide to MF9 was displaced by human αTGF but not by EGF. Bovine pituitary αTGF also displaced the binding of 125I-peptide to MF9 in a similar manner to human αTGF. Both iodinated human and bovine pituitary αTGF were immunoprecipitated by MF9 whereas 125I-EGF was not. Tryptic digests of both 125I-αTGFs chromatographed to give a single, indistinguishable peak of iodinated material on a reverse-phase C18 high performance liquid chromatography column when eluted with two different solvent systems, suggesting the generation of a single and identical tyrosine-containing tryptic peptide from both αTGFs. The comparisons of the bovine pituitary and human melanoma αTGF using a sequence-specific monoclonal antibody and peptide mapping suggest that these αTGFs are related and that αTGF production is not limited to transformed or fetal sources

  20. Gene expression profiling bovine ovarian follicular and luteal cells provides insight into cellular identities and functions

    Science.gov (United States)

    After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...

  1. Transcriptomic analysis of responses to cytopathic bovine viral diarrhea virus-1 (BVDV-1) infection in MDBK cells.

    Science.gov (United States)

    Villalba, Melina; Fredericksen, Fernanda; Otth, Carola; Olavarría, Víctor

    2016-03-01

    The bovine viral diarrhea virus (BVDV) is responsible for significant economic losses in the dairy and cattle industry; however, little is known about the protective and pathological responses of hosts to infection. The present study determined the principal molecular markers implicated in viral infection through meta-transcriptomic analysis using MDBK cells infected for two hours with a field isolate of BVDV-1. While several immune regulator genes were induced, genes involved in cell signaling, metabolic processes, development, and integrity were down-regulated, suggesting an isolation of infected cells from cell-to-cell interactions and responses to external signals. Analysis through RT-qPCR confirmed the expression of more than one hundred markers. Interestingly, there was a significant up-regulation of two negative NF-κB regulators, IER3 and TNFAIP3, indicating a possible blocking of this signaling pathway mediated by BVDV-1 infection. Additionally, several genes involved in the metabolism of reactive oxygen species were down-regulated, suggesting increased oxidative stress. Notably, a number of genes involved in cellular growth and development were also regulated during infection, including MTHFD1L, TGIF1, and Brachyury. Moreover, there was an increased expression of the genes β-catenin, caprin-2, GSK3β, and MMP-7, all of which are crucial to the Wnt signaling pathway that is implicated in the embryonic development of a variety of organisms. This meta-transcriptomic analysis provides the first data towards understanding the infection mechanisms of cytopathic BVDV-1 and the putative molecular relationship between viral and host components. PMID:26919728

  2. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek;

    2009-01-01

    , somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...

  3. Platelet Lysate as Replacement for Fetal Bovine Serum in Mesenchymal Stromal Cell Cultures

    OpenAIRE

    Bieback, Karen

    2013-01-01

    Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in m...

  4. Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages.

    Science.gov (United States)

    Günther, Juliane; Czabanska, Anna; Bauer, Isabel; Leigh, James A; Holst, Otto; Seyfert, Hans-Martin

    2016-01-01

    Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis. PMID:26738804

  5. Investigation of risk factors of bovine mastitis in Ethiopia; Isolation of mastitis causing agents and determination of the content of somatic cells in milk

    OpenAIRE

    Frese, Mathias Lutz

    2010-01-01

    In this thesis, the risk factors of bovine mastitis in different milk production systems in Ethiopia were investigated. Furthermore, mastitis causing agents were isolated after California Mastitis Test (CMT) was used as the field test. Somatic cells were counted and compared with the CMT. Low milk production and low quality of milk are apparently related to a lack of proper hygienic measures throughout the farm clusters.

  6. Patterns of cell proliferation and apoptosis by topographic region in normal Bos taurus vs. Bos indicus crossbreeds bovine placentae during pregnancy

    Directory of Open Access Journals (Sweden)

    Ambrósio Carlos E

    2009-03-01

    Full Text Available Abstract Background Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities. Methods In this study, the proliferation activity and apoptosis in different regions of normal bovine placenta (central and boundary regions of placentomes, placentomal fusion, microplacentomes, and interplacentomal regions, from distinct gestation periods (Days 70 to 290 of pregnancy, were analyzed by flow cytometry. Results Our results indicated that microplacentomes presented a lower number of apoptotic cells throughout pregnancy, with a higher proliferative activity by the end of gestation, suggesting that such structures do not contribute significantly to normal of placental functions and conceptus development during pregnancy. The placentome edges revealed a higher number of apoptotic cells from Day 170 on, which suggests that placentome detachment may well initiate in this region. Conclusion Variations involving proliferation and apoptotic rates may influence placental maturation and detachment, compromising placental functions and leading to fetal stress, abnormalities in development and abortion, as frequently seen in bovine pregnancies from in vitro fertilization and cloning procedures. Our findings describing the pattern of cell proliferation and apoptosis in normal bovine pregnancies may be useful for unraveling some of the developmental deviations seen in nature and after in vitro embryo manipulations.

  7. Effective formation of the segregation-competent complex determines successful partitioning of the bovine papillomavirus genome during cell division.

    Science.gov (United States)

    Silla, Toomas; Männik, Andres; Ustav, Mart

    2010-11-01

    Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4. PMID:20810736

  8. Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization

    Science.gov (United States)

    Medina-Estrada, Ivan; López-Meza, Joel E.

    2016-01-01

    17β-Estradiol (E2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (IIR), and changes in its levels at parturition are related to intramammary infections, such as mastitis. In bovine mammary epithelial cells (bMECs), E2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. Staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bMECs. The aim of this work was to analyze whether E2 modulates the IIR of bMECs during S. aureus internalization. bMECs treated with E2 (50 pg/mL, 24 h) reduced bacteria internalization (~50%). The host receptors α5β1 and TLR2 do not participate in this reduction. However, E2 activates ERα and modulates the IIR reducing the S. aureus induced-mRNA expression of TNF-α (~50%) and IL-1β (90%). E2 also decreased the secretion of these cytokines as well as IL-6 production; however, in infected bMECs, E2 induced the secretion of IL-1β. Furthermore, E2 upregulates the expression of the antimicrobial peptides DEFB1, BNBD5, and psoriasin S100A7 (~5-, 3-, and 6-fold, resp.). In addition, E2 induced the production of antimicrobial compounds in bMEC culture medium, which, together with the modulation of the IIR, could be related to the reduction of S. aureus internalization. PMID:27034592

  9. Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

    Science.gov (United States)

    Gupta, V; Mishra, N; Pateriya, A; Behera, S P; Rajukumar, K

    2014-01-01

    The aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case.

  10. Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets

    Energy Technology Data Exchange (ETDEWEB)

    Fell, A.H.; Preston, P.M. (Edinburgh Univ. (United Kingdom))

    1992-07-01

    Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. (Author).

  11. Molecular Mechanism of Bovine Trabecular Meshwork Cells Apoptosis Induced by Dexamethasone and Protection by Pilocarpine

    Institute of Scientific and Technical Information of China (English)

    Yajuan Gu; Shujun Zeng; Pengxin Qiu; Yuping Wu; Dawei Peng; Guangmei Yan

    2005-01-01

    Purpose: To study the molecular mechanism of trabecular meshwork cells apoptosis induced by dexamethasone and the protection of pilocarpine.Methods: Determining mRNA expression with reverse transcription-polymerase chain reaction (RT-PCR), protein expression with Western blots and the percentage of apoptotic cells with fluorescent microscopy.Results: Dexamethasone up-regulated Fas proteins and affected Bax, caspase-8 and caspase-9 proteins in an action of first decrease then increase. Pre-treatment with pilocarpine decreased the four proteins expression, which were increased by dexamethasone. Pilocarpine self could decrease pro-apoptotic factors Bax, caspase-8 and caspase-9 proteins expression.Conclusion: Fas/FasL pathway participated in apoptotic process induced by dexamethasone in trabecular meshwork cells and the process was probably related with both caspase-8 and caspase-9 pathways. Pilocarpine protected the cells against apoptosis through down-regulating Fas, Bax, caspase-8 and caspase-9 proteins expression.

  12. Expression of Ovine Herpesvirus -2 Encoded MicroRNAs in an Immortalised Bovine - Cell Line

    OpenAIRE

    Katie Nightingale; Levy, Claire S.; John Hopkins; Finn Grey; Suzanne Esper; Dalziel, Robert G.

    2014-01-01

    Ovine herpesvirus-2 (OvHV-2) infects most sheep, where it establishes an asymptomatic, latent infection. Infection of susceptible hosts e.g. cattle and deer results in malignant catarrhal fever, a fatal lymphoproliferative disease characterised by uncontrolled lymphocyte proliferation and non MHC restricted cytotoxicity. The same cell populations are infected in both cattle and sheep but only in cattle does virus infection cause dysregulation of cell function leading to disease. The mechanism...

  13. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    Science.gov (United States)

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  14. Behaviour of oleic acid-depleted bovine alpha-lactalbumin made LEthal to tumor cells (BAMLET).

    Science.gov (United States)

    Hoque, Mehboob; Gupta, Jyoti; Rabbani, Gulam; Khan, Rizwan Hasan; Saleemuddin, M

    2016-05-24

    Oleic acid (OA) complexes of human alpha-lactalbumin (α-LA) and several other proteins are effective in the killing of a variety of tumor cells. While debate on whether the key component of the complexes is the OA or protein continues, studies probing the mechanism of action of the complexes at the tumor cell surface or in the cell interior assume the action of a molecule in the form of an undissociated complex. Recent evidence however suggests that OA complexes of protein are stripped of bound OA on interaction with artificial or natural membranes before entering the cell. Properties of BAMLET stripped of its OA by exposure to erythrocytes (ET-BAMLET) were investigated in the study. ET-BAMLET resembled α-LA in its inability to induce hemolysis of erythrocytes and behaviour in a gel filtration column. Spectroscopy techniques-fluorescence, far- and near UV CD as well as calorimetry and proteolysis however suggest the molecule to be different both from native α-LA and the apo form. Remarkably, unlike native α-LA and apo-α-LA, ET-BAMLET binds OA and turns hemolytic by simple mixing with the fatty acid around neutral pH. Since BAMLET/HAMLET incubated cells take up large amounts of OA, the study suggests the possibility of ET-BAMLET combining with OA and reforming the complex inside the cells. PMID:27109252

  15. Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency.

    Science.gov (United States)

    Kallingappa, Prasanna K; Turner, Pavla M; Eichenlaub, Michael P; Green, Andria L; Oback, Fleur C; Chibnall, Alice M; Wells, David N; Oback, Björn

    2016-07-01

    Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability. PMID:27281704

  16. Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

    Directory of Open Access Journals (Sweden)

    Piccinato Carla A

    2012-11-01

    Full Text Available Abstract Background Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE in the steroid hormone profile of a serum-free granulosa cell (GC culture system in the context of follicular development and dominance. Methods Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose–response study. The highest tested concentration of NE (10 (−7 M resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (−8 M, a non-selective beta-adrenergic antagonist. Conclusions The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

  17. Comparison of the sodium dependency of uptake of meta-lodobenzylguanidine and norepinephrine into cultured bovine adrenomedullary cells

    International Nuclear Information System (INIS)

    Radioiodinated meta-iodobenzylguanidine (MIBG), a scintigraphic agent used for the detection of human pheochromocytomas, is thought to utilize the same uptake and retention mechanism(s) as norepinephrine (NE). Using cultured bovine adrenomedullary cells, the authors compared the mechanism(s) of uptake of MIBG to that of NE. Two different uptake systems were identified. NE and MIBG were taken up by a sodium-dependent system that was characterized by: 1) temperature dependency; 2) high affinity: Km of 1.22 +/- 0.12 microM for MIBG and 1.41 +/- 0.50 microM for NE; 3) low capacity: Vm (picomoles/10(6) cells/10 min) of 64.3 +/- 3.3 for MIBG and 36.6 +/- 7.2 for NE; 4) saturability; 5) ouabain sensitivity; and 6) energy dependency. However, NE and MIBG also were taken up by a temperature-dependent, sodium-independent, apparently unsaturable, and energy-independent system. The sodium-dependent uptake system fulfills many of the criteria for Uptake1 whereas the sodium-independent uptake system is most likely a passive diffusion process. NE uptake proceeded predominantly by the sodium-dependent process. Uptake of MIBG occurred by both pathways at low concentrations, but at high concentrations (greater than 10 microM) uptake was predominantly (75 to 100%) by the sodium-independent process. Inhibition studies suggest that MIBG and NE are transported by the same carrier involved in the sodium-dependent system. Scintiscans of the human adrenals and pheochromocytomas appear to reflect uptake of [131I]MIBG by the sodium-dependent system

  18. Experimental infection with non-cytopathic bovine viral diarrhea virus 1 in mice induces inflammatory cell infiltration in the spleen.

    Science.gov (United States)

    Han, Yu-Jung; Kwon, Young-Je; Lee, Kyung-Hyun; Choi, Eun-Jin; Choi, Kyoung-Seong

    2016-09-01

    Previously, our study showed that oral inoculation of mice with cytopathic (cp) bovine viral diarrhea virus (BVDV) led to lymphocyte depletion and increased numbers of megakaryocytes in the spleen as well as thrombocytopenia and lymphopenia. In the present study, to investigate the possible differences in the detection of viral antigen, histopathological lesions, and hematologic changes between non-cytopathic (ncp) BVDV1 and cp BVDV1, mice were orally administered low and high doses of ncp BVDV1 and were necropsied at days 0, 2, 5, and 9 postinfection (pi). None of the ncp BVDV1-infected mice exhibited clinical signs of illness, unlike those infected with cp BVDV1. Statistically significant thrombocytopenia was observed during ncp BVDV1 infection, and lymphopenia was found only in mice infected with a high dose at day 9 pi. Interestingly, ncp BVDV1 infection increased the numbers of basophils, eosinophils, neutrophils, and monocytes in some infected mice. Viral antigen was detected in the lymphocytes of the spleen, mesenteric lymph nodes, Peyer's patches, and bone marrow by immunohistochemistry. Lymphoid depletion was evident in the mesenteric lymph nodes of mice infected with a high dose and also found in the Peyer's patches of some infected mice. Infiltration of inflammatory cells, including neutrophils and monocytes, and an increased number of megakaryocytes were seen in the spleen. These results suggest that the distribution of viral antigens is not associated with the presence of histopathological lesions. Inflammatory cell infiltration was observed in the spleens as a result of viral replication and may be attributable to the host reaction to ncp BVDV1 infection. Together, these findings support the possibility that mice can be used as an animal model for BVDV infection.

  19. Inhibition of EHMT2 Induces a Robust Antiviral Response Against Foot-and-Mouth Disease and Vesicular Stomatitis Virus Infections in Bovine Cells.

    Science.gov (United States)

    Singh, Neetu; Ramĩrez-Carvajal, Lisbeth; de Los Santos, Teresa; Golding, Michael C; Long, Charles R

    2016-01-01

    The genetic regulatory network controlling the innate immune system is well understood in many species. However, the role of the epigenetic mechanisms underlying the expression of immunoregulatory genes is less clear, especially in livestock species. Histone H3 lysine 9 dimethylation (H3K9me2) is an epigenetic modification associated with transcriptional silencing within the euchromatin regions. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) is a crucial enzyme responsible for regulating the dynamics of this epigenetic modification. It has been shown that histone modifications play a role in regulating type I interferon (IFN) response. In the present study, we investigated the role of EHMT2 in the epigenetic regulation of bovine antiviral innate immunity and explored its therapeutic potential against viral infections. We evaluated the effects of pharmacological and RNAi-mediated inhibition of EHMT2 on the transcription of IFN-β and other IFN-inducible antiviral genes, as well as its effect on foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) replication in bovine cells. We show that treatment of primary bovine cells with the synthetic EHMT2 inhibitor (UNC0638) either before or shortly after virus infection resulted in a significant increase in transcript levels of bovine IFN-β (boIFN-β; 300-fold) and other IFN-inducible genes, including IFN-stimulated gene 15 (ISG-15), myxovirus resistance 1 (Mx-1), Mx-2, RIG-I, 2',5'-oligoadenylate synthetase 1 (OAS-1), and protein kinase R (PKR). Expression of these factors correlated with a significant decrease in VSV and FMDV viral titers. Our data confirm the involvement of EHMT2 in the epigenetic regulation of boIFN-β and demonstrate the activation of a general antiviral state after EHMT2 inhibition. PMID:26418342

  20. Evolution of the CD163 family and its relationship to the bovine gamma delta T cell co-receptor WC1

    Directory of Open Access Journals (Sweden)

    Baldwin Cynthia L

    2010-06-01

    Full Text Available Abstract Background The scavenger receptor cysteine rich (SRCR domain is an ancient and conserved protein domain. CD163 and WC1 molecules are classed together as group B SRCR superfamily members, along with Spα, CD5 and CD6, all of which are expressed by immune system cells. There are three known types of CD163 molecules in mammals, CD163A (M130, coded for by CD163, CD163b (M160, coded for by CD163L1 and CD163c-α (CD163L1 or SCART, while their nearest relative, WC1, is encoded by a multigene family so far identified in the artiodactyl species of cattle, sheep, and pigs. Results We annotated the bovine genome and identified genes coding for bovine CD163A and CD163c-α but found no evidence for CD163b. Bovine CD163A is widely expressed in immune cells, whereas CD163c-α transcripts are enriched in the WC1+ γδ T cell population. Phylogenetic analyses of the CD163 family genes and WC1 showed that CD163c-α is most closely related to WC1 and that chicken and platypus have WC1 orthologous genes, previously classified as among their CD163 genes. Conclusion Since it has been shown that WC1 plays an important role in the regulation of γδ T cell responses in cattle, which, like chickens, have a high percentage of γδ T cells in their peripheral blood, CD163c-α may play a similar role, especially in species lacking WC1 genes. Our results suggest that gene duplications resulted in the expansion of CD163c-α-like and WC1-like molecules. This expanded repertoire was retained by species known as "γδ T cell high", but homologous SRCR molecules were maintained by all mammals.

  1. Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures.

    Science.gov (United States)

    Lemley, C O; Wilson, M E

    2010-10-01

    cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.

  2. Developmental kinetics of the first cell cycles of bovine in vitro PRODUCED EMBRYOS IN RELATION TO THEIR IN VITRO VIABILITY AND SEX

    DEFF Research Database (Denmark)

    Holm, P; Shukri, N.N; Vajta, Gabor;

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g...

  3. Proliferation rates of bovine primary muscle cells relate to liveweight and carcase weight in cattle.

    Science.gov (United States)

    Coles, Chantal A; Wadeson, Jenny; Leyton, Carolina P; Siddell, Jason P; Greenwood, Paul L; White, Jason D; McDonagh, Matthew B

    2015-01-01

    Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5-20 hours in culture) in vitro (P cattle (P cattle (P cattle.

  4. Role of peroxidase inhibition by insulin in the bovine thyroid cell proliferation mechanism.

    Science.gov (United States)

    Krawiec, León; Pizarro, Ramón A; Aphalo, Paula; de Cavanagh, Elena M V; Pisarev, Mario A; Juvenal, Guillermo J; Policastro, Lucía; Bocanera, Laura V

    2004-07-01

    Monolayer primary cultures of thyroid cells produce, in the presence of insulin, a cytosolic inhibitor of thyroid peroxidase (TPO), lacto peroxidase (LPO), horseradish peroxidase (HRPO) and glutathione peroxidase (GPX). The inhibitor, localized in the cytosol, is thermostable and hydrophylic. Its molecular mass is less than 2 kDa. The inhibitory activity, resistant to proteolytic and nucleolytic enzymes, disappears with sodium metaperiodate treatment, as an oxidant of carbohydrates, supporting its oligosaccharide structure. The presence of inositol, mannose, glucose, the specific inhibition of cyclic AMP-dependent protein kinase and the disappearance of peroxidase inhibition by alkaline phosphatase and alpha-mannosidase in purified samples confirms its chemical structure as inositol phosphoglycan-like. Purification by anionic interchange shows that the peroxidase inhibitor elutes like the two subtypes of inositol phosphoglycans (IPG)P and A, characterized as signal transducers of insulin action. Insulin significantly increases the concentration of the peroxidase inhibitor in a thyroid cell culture at 48 h. The addition of both isolated substances to a primary thyroid culture produces, after 30 min, a significant increase in hydrogen peroxide (H2O2) concentration in the medium, concomitantly with the disappearance of the GPX activity in the same conditions. The presence of insulin or anyone of both products, during 48 h, induces cell proliferation of the thyroid cell culture. In conclusion, insulin stimulates thyroid cell division through the effect of a peroxidase inhibitor, as its second messenger. The inhibition of GPX by its action positively modulates the H2O2 level, which would produce, as was demonstrated by other authors, the signal for cell proliferation.

  5. Nutrient-induced modulation of gene expression and cellular functions: modeling epigenetic regulation in bovine cells

    Science.gov (United States)

    Volatile fatty acids (VFA), especially butyrate, participate in metabolism both as nutrients and as regulators of histone deacetylation. The major biochemical change that occurs in cells treated with butyrate is the global hyperacetylation of histones. One paradigmatic example of the nutrient-epige...

  6. Longitudinal characterization of bovine monocyte-derived dendritic cells from mid-gestation into subsequent lactation reveals nadir in phenotypic maturation and macrophage-like cytokine profile in late gestation

    NARCIS (Netherlands)

    Pomeroy, Brianna; Sipka, Anja; Klaessig, Suzanne; Schukken, Ynte

    2016-01-01

    Changes in monocyte and dendritic cell populations during bovine pregnancy and lactation remain poorly described despite the key roles these cells play in immune tolerance and activation. Using a prospective longitudinal study, we characterized CD14+ monocyte-derived dendritic cell (moDC) differenti

  7. Osteoclast-like cells on deproteinized bovine bone mineral and biphasic calcium phosphate

    DEFF Research Database (Denmark)

    Jensen, Simon S; Gruber, Reinhard; Buser, Daniel;

    2015-01-01

    OBJECTIVES: The occurrence of multinucleated giant cells (MNGCs) on bone substitute materials has been recognized for a long time. However, there have been no studies linking material characteristics with morphology of the MNGCs. The aim was to analyze the qualitative differences of MNGCs on two...... microscopy. RESULTS: Multinucleated giant cells appeared on both biomaterials. On BCP, MNGCs had a flat morphology and were not observed in resorption lacunae. On DBBM, the MNGCs appeared more round and were often found in shallow concavities. MNGCs on both biomaterials demonstrated a varying degree of TRAP...... osteoclasts. CONCLUSION: MNGCs demonstrated distinctly different histological features depending on the bone substitute material used. Further research is warranted to understand the clinical implications of these morphological observations....

  8. Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique

    DEFF Research Database (Denmark)

    Booth, P J; Tan, S J; Reipurth, R;

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods......, and (c) establish any potential embryotoxic effects of PHA-P. The initial data indicated that, of calcium ionophore A23187, ionomycin, and electropulse treatments as primary activation agents, the two former were equally efficient even with reduced exposure times. WOW-culture of zona-free versus zona.......8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1 blastocysts per successfully fused and surviving reconstructed...

  9. Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

    OpenAIRE

    McLaughlin, Kerry; Seago, Julian; Robinson, Lucy; Kelly, Charles; Charleston, Bryan

    2010-01-01

    International audience Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4+ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to th...

  10. Regulation of Major Histocompatibility Complex Class I Genes in Bovine Trophoblast Cells

    OpenAIRE

    Shi, Bi

    2014-01-01

    Somatic cell nuclear transfer (SCNT), or cloning, is a form of artificial reproductive technology that can be used to improve economic traits of domestic animals. However, extreme inefficiency of producing viable offspring via this method is a major limitation. An aggressive immune response at the maternal-fetal interface is an important reason for SCNT pregnancy loss. The goal of this project was to investigate the molecular mechanisms of immune-mediated miscarriage in cloned cattle pregnanc...

  11. In vitro effects of the organochlorine pesticide β-hexachlorocyclohexane on bovine peripheral blood mononuclear cells

    OpenAIRE

    Cristina Rossi; Pier Paolo Danieli; Bruno Ronchi

    2014-01-01

    The β-hexachlorocyclohexane (β-HCH) is a very stable and accumulable isomer of Lindane, a well known organochlorine pesticide. The HCHs were banned in all developed countries but to date high concern still exists for environment, animal and human health due to contaminated sites. In this study, several in vitro tests [cell viability (XTT), trypan blue exclusion (TBE), lactate dehydrogenase release (LDH) and bromodeoxyuridine (BrdU) incorporation assays] were performed to investigate the toxic...

  12. Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

    DEFF Research Database (Denmark)

    Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe;

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... staining for detection of apoptotic nuclear morphology, and subjected to fluorescence microscopy. Additionally, treated and untreated blastocysts were fixed and processed for ultrastructural identification of apoptosis. Untreated embryos revealed no apoptotic features at 2- and 4-cell stages. However......, active caspase-3 and apoptotic nuclear morphology were observed in an untreated 8-cell stage, and TUNEL-labeling was observed from the 16-cell stage. Blastomeres concurrently displaying all apoptotic features were present in a few embryos at 16-cell and morula stages and in all blastocysts. All three...

  13. Antiviral effects of bovine interferons on bovine respiratory tract viruses.

    OpenAIRE

    Fulton, R W; Downing, M M; Cummins, J M

    1984-01-01

    The antiviral effects of bovine interferons on the replication of bovine respiratory tract viruses were studied. Bovine turbinate monolayer cultures were treated with bovine interferons and challenged with several bovine herpesvirus 1 strains, bovine viral diarrhea virus, parainfluenza type 3 virus, goat respiratory syncytial virus, bovine respiratory syncytial virus, bovine adenovirus type 7, or vesicular stomatitis virus. Treatment with bovine interferons reduced viral yield for each of the...

  14. Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies

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    Laming Eleanor

    2012-10-01

    Full Text Available Abstract Background Bovine neonatal pancytopenia (BNP is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows (“BNP dams”. Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and β-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. Results Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage was markedly reduced. Conclusion This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

  15. The Neuroprotective Potential of Rho-Kinase Inhibition in Promoting Cell Survival and Reducing Reactive Gliosis in Response to Hypoxia in Isolated Bovine Retina

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    Aizhan Alt

    2013-07-01

    Full Text Available Aims: To investigate the outcomes of Rho-kinase inhibition in the electrophysiological ex vivo model of the isolated perfused vertebrate retina under hypoxia. Methods: Bovine retinas were perfused with an oxygen saturated nutrient solution with or without the Rho-kinase inhibitor H-1152P. The retinas were stimulated repeatedly until stable amplitudes were reached and the electroretinogram was recorded at five minute intervals. Hypoxia was induced for 15, 30, and 45 minutes, after which the oxygen saturation was restored. The extent of the cell damage and glial reactivity was determined by Ethidium homodimer-1 staining, immunohistochemistry, and Western blot. Results: Hypoxia caused a time-dependent reduction of the b-wave amplitudes, which could not be prevented by the H-1152P. Although the Rho-kinase inhibitor maintained higher b-wave amplitudes, these effects did not reach statistical significance. Hypoxia also resulted in an increase in cell damage and the activation of the glial cells in the untreated retinas whereas the administration of H-1152P significantly reduced the extent of these events. Conclusion: H-1152P exerted a neuroprotective effect against necrosis on the isolated bovine retina under hypoxia together with a reduction in glial cell reactivity. However, the inhibitor could not prevent the hypoxia induced retinal dysfunction possibly due to the interference with synaptic modulation.

  16. Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

    Science.gov (United States)

    Murakami, K; Uchiyama, A; Kokuho, T; Mori, Y; Sentsui, H; Yada, T; Tanigawa, M; Kuwano, A; Nagaya, H; Ishiyama, S; Kaki, H; Yokomizo, Y; Inumaru, S

    2001-01-01

    The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells. PMID:11145838

  17. Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation.

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    Isabelle Hue

    Full Text Available In addition to nourishing the embryo, extra-embryonic tissues (EETs contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC, endoderm (bXEC, and mesoderm (bXMC cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify "cores" of cell-type-specific (and substrate-independent genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21-D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in

  18. Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division ▿ †

    OpenAIRE

    Silla, Toomas; Männik, Andres; Ustav, Mart

    2010-01-01

    Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral geno...

  19. Activation of cell signaling pathways is dependant on the biotype of bovine viral diarrhea virus type 2

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a world wide distribution. Besides the segregation into two distinct species (BVDV1 / BVDV2) two different biotypes, a cytopathic (cp) and a noncytopathic (ncp) biotype, are...

  20. A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

    Directory of Open Access Journals (Sweden)

    Ustav Mart

    2002-04-01

    Full Text Available Abstract Background The rationale of using bovine papillomavirus-1 (BPV-1 derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. Results The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Conclusion Bovine papillomavirus type-1 (BPV-1-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

  1. Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells.

    OpenAIRE

    Shimada, K.; Gill, P J; Silbert, J E; Douglas, W H; Fanburg, B L

    1981-01-01

    It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the inter...

  2. A Continuous Bovine Kidney Cell Line Constitutively Expressing Bovine αVβ6 Integrin Has Increased Susceptibility to Foot-and-Mouth Disease Virus

    OpenAIRE

    LaRocco, Michael; Krug, Peter W; Kramer, Ed; Ahmed, Zaheer; Pacheco, Juan M.; Duque, Hernando; Baxt, Barry; Luis L Rodriguez

    2013-01-01

    Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and mainta...

  3. Bovine Tuberculosis, A Zoonotic Disease

    Directory of Open Access Journals (Sweden)

    Tarmudji

    2008-12-01

    Full Text Available Bovine tuberculosis is caused by the infection of Mycobacterium tuberculosis var. bovis (M. bovis. This species is one of Mycobacterium tuberculosis complex, can infect wide range of hosts: cattle and other domesticated animals, wild mammals and humans (zoonotic. M. bovis bacterium from infected hosts can be transmitted to other susceptible animals and humans through respiratory excretes and secretion materials. Humans can be infected with M. bovis by ingested M. bovis contaminated animal products, unpasteurised milk from tuberculosis cows or through respiratory route of contaminated aerosol. Bovine tuberculosis at the first stage does not show any clinical sign but as the disease progress in the next stage which may take several months or years, clinical signs may arise, suh as: fluctuative body temperature, anorexia, lost body weight, coughing, oedema of lymph nodes, increased respiratory frequencies. Pathological lesion of bovine tuberculosis is characterised by the formation of granulomas (tubercles, in which bacterial cells have been localised, most in lymph nodes and pulmonum, but can occur in other organs. The granulomas usually arise in small nodules or tubercles appear yellowish either caseus, caseo-calcareus or calcified. In Indonesia, bovine tuberculosis occurred in dairy cattle since 1905 through the imported dairy cows from Holland and Australian. It was unfortunate that until recently, there were not many research and surveilances of bovine tuberculosis conducted in this country, so the distribution of bovine tuberculosis is unknown. Early serological diagnosis can be done on live cattle by means of tuberculin tests under field conditions. Confirmation can be done by isolation and identification of excreted and secreted samples from the slaughter house. Antibiotic treatment and vaccination were uneffective, therefore the effective control of bovine tuberculosis is suggested by tuberculin tests and by slaughtering the selected

  4. Relationship between the length of cell cycles, cleavage pattern and developmental competence in bovine embryos generated by in vitro fertilization or parthenogenesis.

    Science.gov (United States)

    Somfai, Tamás; Inaba, Yasushi; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Shuji; Konishi, Kazuyuki; Imai, Kei

    2010-04-01

    This study was conducted to study the kinetics of initial cell divisions in relation with the cleavage patterns in viable (with the ability to develop to the blastocyst stage) and non-viable bovine embryos and parthenotes. The kinetics of in vitro development and cleavage patterns were observed by time lapse cinematography. The length of the first and second but not third cell cycle differed significantly between the viable and non-viable embryos after IVF or parthenogenesis. Viable embryos had significantly shorter first and second cell cycles than non-viable ones. The presence of fragments, protrusions and unequally-sized blastomeres was associated with an extended one-cell stage and reduced ability to develop to the blastocyst stage; however, the lengths of the second and third cell cycles were not altered. Oocytes showing direct division from one cell to 3 or 4 blastomeres showed similar developmental ability and embryonic cell numbers to those showing normal division, although, with a high frequency of chromosomal abnormalities. Our results suggest that the differences in the first cell cycles between viable and non-viable embryos were not sperm-related, whereas direct cleavage of 1-cell embryos to 3 or more blastomeres and protrusion formation are related to sperm-driven factors. The length of the first and second cell cycles and the cleavage pattern should be examined simultaneously to predict developmental competence of embryos at early cleavage stages. PMID:20035110

  5. Identification of bovine leukemia virus tax function associated with host cell transcription, signaling, stress response and immune response pathway by microarray-based gene expression analysis

    Directory of Open Access Journals (Sweden)

    Arainga Mariluz

    2012-03-01

    Full Text Available Abstract Background Bovine leukemia virus (BLV is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type I. The Tax protein of BLV is a transcriptional activator of viral replication and a key contributor to oncogenic potential. We previously identified interesting mutant forms of Tax with elevated (TaxD247G or reduced (TaxS240P transactivation effects on BLV replication and propagation. However, the effects of these mutations on functions other than transcriptional activation are unknown. In this study, to identify genes that play a role in the cascade of signal events regulated by wild-type and mutant Tax proteins, we used a large-scale host cell gene-profiling approach. Results Using a microarray containing approximately 18,400 human mRNA transcripts, we found several alterations after the expression of Tax proteins in genes involved in many cellular functions such as transcription, signal transduction, cell growth, apoptosis, stress response, and immune response, indicating that Tax protein has multiple biological effects on various cellular environments. We also found that TaxD247G strongly regulated more genes involved in transcription, signal transduction, and cell growth functions, contrary to TaxS240P, which regulated fewer genes. In addition, the expression of genes related to stress response significantly increased in the presence of TaxS240P as compared to wild-type Tax and TaxD247G. By contrast, the largest group of downregulated genes was related to immune response, and the majority of these genes belonged to the interferon family. However, no significant difference in the expression level of downregulated genes was observed among the Tax proteins. Finally, the expression of important cellular factors obtained from the human microarray results were validated at the RNA and protein levels by real-time quantitative reverse transcription-polymerase chain reaction and western blotting

  6. Occurrence of a cytosolic neutral chitobiase activity involved in oligomannoside degradation: a study with Madin-Darby bovine kidney (MDBK) cells.

    Science.gov (United States)

    Cacan, R; Dengremont, C; Labiau, O; Kmiécik, D; Mir, A M; Verbert, A

    1996-01-15

    Neutral oligomannosides possessing one GlcNAc (OS-Gn1) and two GlcNAc (Os-Gn2) at the reducing end have been reported to be released during the N-glycosylation process in various biological models. To investigate which enzyme is responsible for OS-Gn1 formation, we used the Madin-Darby bovine kidney (MDBK) cell line which exhibits neither lysosomal chitobiase nor endoglucosaminidase activities. However, these cells produced OS-Gn1 and we showed that a neutral chitobiase is responsible for the transformation of OS-Gn2 into OS-Gn1. Using streptolysin O-permeabilized MDBK cells, we demonstrated that this neutral chitobiase activity is located in the cytosolic compartment and is active on oligomannoside species released during the N-glycosylation process.

  7. Kinetics of adrenal medullary cells.

    OpenAIRE

    Verhofstad, A A

    1993-01-01

    The adrenal medulla of mammals has a heterogeneous population of cells. In adults most are epithelial cells containing a particular type of cytoplasmic granule. Based on a variety of cytochemical and ultrastructural studies it is now accepted that 2 different adrenal medullary chromaffin cell types can be distinguished, i.e. noradrenaline (NA) and adrenaline (A) synthesising and storing cells. Other cell types present in the adrenal medulla include neuronal elements comprising either cell bod...

  8. Functional Interactions between 17β-Estradiol and Progesterone Regulate Autophagy during Acini Formation by Bovine Mammary Epithelial Cells in 3D Cultures

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    Katarzyna Zielniok

    2014-01-01

    Full Text Available Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2 and progesterone (P4. Mammary epithelial cells (MECs cultured on reconstituted basement membrane (rBM form three-dimensional (3D acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR involved in autophagy induction.

  9. A naturally occurring α(s1)-casein-derived peptide in bovine milk inhibits apoptosis of granulosa cells induced by serum-free conditions.

    Science.gov (United States)

    Shimizu, T; Ganzorig, K; Miyamoto, A; Ishii, T; Urashima, T; Fukuda, K

    2014-03-01

    Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra-filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α(s1)-CN (f16-23) (RPKHPIKH), α(s1)-CN (f16-24) (RPKHPIKHQ), α(s1)-CN (f17-25) (PKHPIKHQG), α(s1)-CN (f46-52) (VFGKEKV), α(s1)-CN (f94-105) (HIQKEDVPSER), and β-CN (f121-128) (HKEMPFPK), were identified. One of the major peptides, the N-terminal fragment of αs1 -casein, varied structurally during early lactation: α(s1)-CN (f17-25) (PKHPIKHQG) and α(s1)-CN (f16-23) (RPKHPIKH)/α(s1)-CN (f16-24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α(s1)-CN (f16-23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum-free conditions in a dose-dependent manner, in consequence of caspase-3 and caspase-9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti-apoptotic agent in cell culture medium. PMID:24399684

  10. Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages.

    Science.gov (United States)

    Everman, Jamie L; Bermudez, Luiz E

    2015-01-01

    Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection. PMID:26301206

  11. The bovine model for elucidating the role of γδ T cells in controlling infectious diseases of importance to cattle and humans.

    Science.gov (United States)

    Baldwin, Cynthia L; Telfer, Janice C

    2015-07-01

    There are several instances of co-investigation and related discoveries and achievements in bovine and human immunology; perhaps most interesting is the development of the BCG vaccine, the tuberculin skin test and the more recent interferon-gamma test that were developed first in cattle to prevent and diagnosis bovine tuberculosis and then applied to humans. There are also a number of immune-physiological traits that ruminant share with humans including the development of their immune systems in utero which increases the utility of cattle as a model for human immunology. These are reviewed here with a particular focus on the use of cattle to unravel γδ T cell biology. Based on the sheer number of γδ T cells in this γδ T cell high species, it is reasonable to expect γδ T cells to play an important role in protective immune responses. For that reason alone cattle may provide good models for elucidating at least some of the roles γδ T cells play in protective immunity in all species. This includes fundamental research on γδ T cells as well as the responses of ruminant γδ T cells to a variety of infectious disease situations including to protozoan and bacterial pathogens. The role that pattern recognition receptors (PRR) play in the activation of γδ T cells may be unique relative to αβ T cells. Here we focus on that of the γδ T cell specific family of molecules known as WC1 or T19 in ruminants, which are part of the CD163 scavenger receptor cysteine rich (SRCR) family that includes SCART1 and SCART2 expressed on murine γδ T cells. We review the evidence for WC1 being a PRR as well as an activating co-receptor and the role that γδ T cells bearing these receptors play in immunity to leptospirosis and tuberculosis. This includes the generation of memory responses to vaccines, thereby continuing the tradition of co-discovery between cattle and humans.

  12. ERas protein is overexpressed and binds to the activated platelet-derived growth factor β receptor in bovine urothelial tumour cells associated with papillomavirus infection.

    Science.gov (United States)

    Russo, Valeria; Roperto, Franco; Esposito, Iolanda; Ceccarelli, Dora Maria; Zizzo, Nicola; Leonardi, Leonardo; Capparelli, Rosanna; Borzacchiello, Giuseppe; Roperto, Sante

    2016-06-01

    Embryonic stem cell-expressed Ras (ERas) encodes a constitutively active form of guanosine triphosphatase (GTPase) that binds to and activates phosphatidylinositol 3 kinase (PI3K), which in turn phosphorylates and activates downstream targets such as Akt. The current study evaluated ERas regulation and expression in papillomavirus-associated urothelial tumours in cattle grazing on lands rich in bracken fern. ERas was found upregulated and overexpressed by PCR, real time PCR and Western blot. Furthermore, protein overexpression was also confirmed by immunohistochemistry. ERas was found to interact physically and colocalise with the activated platelet derived growth factor β receptor (PDGFβR) by coimmunoprecipitation and laser scanning confocal investigations. Phosphorylation of Akt, a downstream effector both of ERas and PDGFβR, appeared to be increased in urothelial tumour cells. Altogether, these data indicate that ERas/PDGFβR complex could play a role in the pathogenesis of bovine papillomavirus-associated bladder neoplasia. PMID:27256024

  13. Proline modulates the effect of bisphosphonate on calcium levels and adenosine triphosphate production in cell lines derived from bovine Echinococcus granulosus protoscoleces.

    Science.gov (United States)

    Fuchs, A G; Echeverría, C I; Pérez Rojo, F G; Prieto González, E A; Roldán, E J A

    2014-12-01

    Bisphosphonates have been proposed as pharmacological agents against parasite and cancer cell growth. The effect of these compounds on helminthic cell viability and acellular compartment morphology, however, has not yet been studied. The effects of different types of bisphosphonates, namely etidronate (EHDP), pamidronate (APD), alendronate (ABP), ibandronate (IB) and olpadronate (OPD), and their interaction with amiloride, 1,25-dihydroxycholecalciferol (D3) and proline were evaluated on a cell line derived from bovine Echinococcus granulousus protoscoleces (EGPE) that forms cystic colonies in agarose. The EGPE cell line allowed testing the effect of bisphosphonates alone and in association with other compounds that could modulate calcium apposition/deposition, and were useful in measuring the impact of these compounds on cell growth, cystic colony formation and calcium storage. Decreased cell growth and cystic colony formation were found with EHDP, IB and OPD, and increased calcium storage with EHDP only. Calcium storage in EGPE cells appeared to be sensitive to the effect of amiloride, D3 and proline. Proline decreased calcium storage and increased colony formation. Changes in calcium storage may be associated with degenerative changes of the cysts, as shown in the in vitro colony model and linked to an adenosine triphosphate (ATP) decrease. In conclusion, bisphosphonates could be suitable tempering drugs to treat cestode infections.

  14. [Construction of recombinant retroviral vector carrying Lab gene of foot-and-mouth disease virus and its expression in bovine kidney (MDBK) cells].

    Science.gov (United States)

    Cong, Guozheng; Zhou, Jianhua; Gao, Shandian; Du, Junzheng; Shao, Junjun; Lin, Tong; Chang, Huiyun; Xie, Qingge

    2008-05-01

    In this study, foot-and-mouth disease virus (FMDV) strain OA/58 RNAs were used as templates for RT-PCR. By the molecular cloning, the Lab gene encoding leader protease called Lpro were cloned in retroviral vector pBPSTR1 to obtain reconstruction retroviral vector termed pBPSTR1-Lab. At different concentrations of puromycin and tetracycline respectively in the cell culture mediums, the growth of bovine kidney cells (MDBK) showed that the optimal puromycin resistant selection concentration was 3 microg/mL and tetracycline regulatory concentration was 1 microg/mL. Pseudotyped retroviral virus particles were produced by transiently co-tansfecting GP2-293 cells with a retroviral vector DNA and VSV-G plasmid. Then MDBK cells were infected by pseudotyped retroviral virus and were continually seeded in the medium at the optimal tetracycline regulatory concentration and puromycin selection concentration for 12 days to obtain puromycin resistant colonies whose genomes contained the Lab gene. After tetracycline removal, synthesis of Lpro induced severe morphological changes in the puromycin resistant MDBK cells. PCR and Western blotting proved that a stable MDBK cell line inducibly expressing the Lab gene under the control of tetracycline was obtained. The experiment might provide a basis for studying that Lpro of FMDV plays an important role in MDBK cell pathogenesis.

  15. Productive infection of bovine papillomavirus type 2 in the urothelial cells of naturally occurring urinary bladder tumors in cattle and water buffaloes.

    Directory of Open Access Journals (Sweden)

    Sante Roperto

    Full Text Available BACKGROUND: Papillomaviruses (PVs are highly epitheliotropic as they usually establish productive infections within squamous epithelia of the skin, the anogenital tract and the oral cavity. In this study, early (E and late (L protein expression of bovine papillomavirus type 2 (BPV-2 in the urothelium of the urinary bladder is described in cows and water buffaloes suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. METHODS AND FINDINGS: E5 protein, the major oncoprotein of the BPV-2, was detected in all tumors. L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection was detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium. Finally, the early protein E2, required for viral DNA replication and known to be a pivotal factor for both productive and persistent infection, was detected by Western blot and immunohistochemically. Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium. CONCLUSION: This study shows that both active and productive infections by BPV-2 in the urothelium of the bovine and bubaline urinary bladder can occur in vivo.

  16. Safety and efficacy of an E2 glycoprotein subunit vaccine produced in mammalian cells to prevent experimental infection with bovine viral diarrhoea virus in cattle.

    Science.gov (United States)

    Pecora, Andrea; Aguirreburualde, María Sol Pérez; Aguirreburualde, Alejandra; Leunda, Maria Rosa; Odeon, Anselmo; Chiavenna, Sebastián; Bochoeyer, Diego; Spitteler, Marcelo; Filippi, Jorge L; Dus Santos, Maria J; Levy, Susana M; Wigdorovitz, Andrés

    2012-09-01

    Bovine viral diarrhea (BVD) infection caused by bovine viral diarrhea virus (BVDV), a Pestivirus of the Flaviviridae family, is an important cause of morbidity, mortality and economical losses in cattle worldwide. E2 protein is the major glycoprotein of BVDV envelope and the main target for neutralising antibodies (NAbs). Different studies on protection against BVDV infection have focused on E2, supporting its putative use in subunit vaccines. A truncated version of type 1a BVDV E2 (tE2) expressed in mammalian cells was used to formulate an experimental oleous monovalent vaccine. Immunogenicity was studied through immunisation of guinea pigs and followed by trials in cattle. Calves of 8-12 months were vaccinated, twice with a 4 week interval, with either a tE2 subunit vaccine (n = 8), a whole virus inactivated vaccine (n = 8) or left untreated as negative control group (n = 8). Four weeks after the last immunisation the animals were experimentally challenged intranasally with a non-cythopathic BVDV strain. Following challenge, BVDV was isolated from all unvaccinated animals, while 6 out of 8 animals vaccinated with tE2 showed complete virological protection indicating that the tE2 vaccine presented a similar performance to a satisfactory whole virus inactivated vaccine.

  17. Regulation of Calcium Channels and Exocytosis in Mouse Adrenal Chromaffin Cells by Prostaglandin EP3 Receptors

    OpenAIRE

    Jewell, Mark L.; Breyer, Richard M.; Currie, Kevin P.M.

    2011-01-01

    Prostaglandin (PG) E2 controls numerous physiological functions through a family of cognate G protein-coupled receptors (EP1–EP4). Targeting specific EP receptors might be therapeutically useful and reduce side effects associated with nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors that block prostanoid synthesis. Systemic immune challenge and inflammatory cytokines have been shown to increase expression of the synthetic enzymes for PGE2 in the adrenal gland. Ca...

  18. Doc2b synchronizes secretion from chromaffin cells by stimulating fast and inhibiting sustained release

    DEFF Research Database (Denmark)

    da Silva Pinheiro, Paulo César; de Wit, Heidi; Walter, Alexander M;

    2013-01-01

    is still high. We conclude that Doc2b acts to inhibit vesicle priming during prolonged calcium elevations, thus protecting unprimed vesicles from fusing prematurely, and redirecting them to refill the readily releasable pool after relaxation of the calcium signal. In sum, Doc2b favors fast, synchronized...

  19. Effects of injectable trace minerals on humoral and cell-mediated immune responses to Bovine viral diarrhea virus, Bovine herpes virus 1 and Bovine respiratory syncytial virus following administration of a modified-live virus vaccine in dairy calves.

    Science.gov (United States)

    Palomares, R A; Hurley, D J; Bittar, J H J; Saliki, J T; Woolums, A R; Moliere, F; Havenga, L J; Norton, N A; Clifton, S J; Sigmund, A B; Barber, C E; Berger, M L; Clark, M J; Fratto, M A

    2016-10-01

    Our objective was to evaluate the effect of an injectable trace mineral (ITM) supplement containing zinc, manganese, selenium, and copper on the humoral and cell mediated immune (CMI) responses to vaccine antigens in dairy calves receiving a modified-live viral (MLV) vaccine containing BVDV, BHV1, PI3V and BRSV. A total of 30 dairy calves (3.5 months of age) were administered a priming dose of the MLV vaccine containing BHV1, BVDV1 & 2, BRSV, PI3V, and an attenuated-live Mannheimia-Pasteurella bacterin subcutaneously (SQ). Calves were randomly assigned to 1 of 2 groups: (1) administration of ITM SQ (ITM, n=15) or (2) injection of sterile saline SQ (Control; n=15). Three weeks later, calves received a booster of the same vaccine combination SQ, and a second administration of ITM, or sterile saline, according to the treatment group. Blood samples were collected on days 0, 7, 14, 21, 28, 42, 56, and 90 post-vaccination for determination of antibody titer, viral recall antigen-induced IFN-γ production, and viral antigen-induced proliferation by peripheral blood mononuclear cells (PBMC). Administration of ITM concurrently with MLV vaccination resulted in higher antibody titers to BVDV1 on day 28 after priming vaccination compared to the control group (P=0.03). Calves treated with ITM showed an earlier enhancement in PBMC proliferation to BVDV1 following vaccination compared to the control group. Proliferation of PBMC after BVDV stimulation tended to be higher on day 14 after priming vaccination in calves treated with ITM than in the control group (P=0.08). Calves that received ITM showed higher PBMC proliferation to BRSV stimulation on day 7 after priming vaccination compared to the control group (P=0.01). Moreover, calves in the ITM group also had an enhanced production IFN-γ by PBMC after stimulation with BRSV on day 21 after priming vaccination compared to day 0 (P<0.01). In conclusion, administration of ITM concurrently with MLV vaccination in dairy calves

  20. Effects of injectable trace minerals on humoral and cell-mediated immune responses to Bovine viral diarrhea virus, Bovine herpes virus 1 and Bovine respiratory syncytial virus following administration of a modified-live virus vaccine in dairy calves.

    Science.gov (United States)

    Palomares, R A; Hurley, D J; Bittar, J H J; Saliki, J T; Woolums, A R; Moliere, F; Havenga, L J; Norton, N A; Clifton, S J; Sigmund, A B; Barber, C E; Berger, M L; Clark, M J; Fratto, M A

    2016-10-01

    Our objective was to evaluate the effect of an injectable trace mineral (ITM) supplement containing zinc, manganese, selenium, and copper on the humoral and cell mediated immune (CMI) responses to vaccine antigens in dairy calves receiving a modified-live viral (MLV) vaccine containing BVDV, BHV1, PI3V and BRSV. A total of 30 dairy calves (3.5 months of age) were administered a priming dose of the MLV vaccine containing BHV1, BVDV1 & 2, BRSV, PI3V, and an attenuated-live Mannheimia-Pasteurella bacterin subcutaneously (SQ). Calves were randomly assigned to 1 of 2 groups: (1) administration of ITM SQ (ITM, n=15) or (2) injection of sterile saline SQ (Control; n=15). Three weeks later, calves received a booster of the same vaccine combination SQ, and a second administration of ITM, or sterile saline, according to the treatment group. Blood samples were collected on days 0, 7, 14, 21, 28, 42, 56, and 90 post-vaccination for determination of antibody titer, viral recall antigen-induced IFN-γ production, and viral antigen-induced proliferation by peripheral blood mononuclear cells (PBMC). Administration of ITM concurrently with MLV vaccination resulted in higher antibody titers to BVDV1 on day 28 after priming vaccination compared to the control group (P=0.03). Calves treated with ITM showed an earlier enhancement in PBMC proliferation to BVDV1 following vaccination compared to the control group. Proliferation of PBMC after BVDV stimulation tended to be higher on day 14 after priming vaccination in calves treated with ITM than in the control group (P=0.08). Calves that received ITM showed higher PBMC proliferation to BRSV stimulation on day 7 after priming vaccination compared to the control group (P=0.01). Moreover, calves in the ITM group also had an enhanced production IFN-γ by PBMC after stimulation with BRSV on day 21 after priming vaccination compared to day 0 (P<0.01). In conclusion, administration of ITM concurrently with MLV vaccination in dairy calves

  1. Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells

    Directory of Open Access Journals (Sweden)

    Roy K

    2015-12-01

    Full Text Available Kislay Roy,1,* Yogesh S Patel,1,* Rupinder K Kanwar,1 Rangam Rajkhowa,2 Xungai Wang,2 Jagat R Kanwar1 1Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, Centre for Molecular and Medical Research (C-MMR, School of Medicine (SoM, Faculty of Health, 2Institute for Frontier Materials (IFM, Deakin University, Waurn Ponds, VIC, Australia *These authors contributed equally to this work Abstract: This study used the Eri silk nanoparticles (NPs for delivering apo-bovine lactoferrin (Apo-bLf (~2% iron saturated and Fe-bLf (100% iron saturated in MDA-MB-231 and MCF-7 breast cancer cell lines. Apo-bLf and Fe-bLf-loaded Eri silk NPs with sizes between 200 and 300 nm (±10 nm showed a significant internalization within 4 hours in MDA-MB-231 cells when compared to MCF-7 cells. The ex vivo loop assay with chitosan-coated Fe-bLf-loaded silk NPs was able to substantiate its future use in oral administration and showed the maximum absorption within 24 hours by ileum. Both Apo-bLf and Fe-bLf induced increase in expression of low-density lipoprotein receptor-related protein 1 and lactoferrin receptor in epidermal growth factor (EGFR-positive MDA-MB-231 cells, while transferrin receptor (TfR and TfR2 in MCF-7 cells facilitated the receptor-mediated endocytosis of NPs. Controlled and sustained release of both bLf from silk NPs was shown to induce more cancer-specific cytotoxicity in MDA-MB-231 and MCF-7 cells compared to normal MCF-10A cells. Due to higher degree of internalization, the extent of cytotoxicity and apoptosis was significantly higher in MDA-MB-231 (EGFR+ cells when compared to MCF-7 (EGFR- cells. The expression of a prominent anti-cancer target, survivin, was found to be downregulated at both gene and protein levels. Taken together, all the observations suggest the potential use of Eri silk NPs as a delivery vehicle for an anti-cancer milk protein, and indicate bLf for the treatment of breast cancer. Keywords: breast

  2. Cytotoxicity and intracellular fate of PLGA and chitosan-coated PLGA nanoparticles in Madin-Darby bovine kidney (MDBK) and human colorectal adenocarcinoma (Colo 205) cells.

    Science.gov (United States)

    Trif, Mihaela; Florian, Paula E; Roseanu, Anca; Moisei, Magdalena; Craciunescu, Oana; Astete, Carlos E; Sabliov, Cristina M

    2015-11-01

    Polymeric nanoparticles (NPs) are known to facilitate intracellular uptake of drugs to improve their efficacy, with minimum bioreactivity. The goal of this study was to assess cellular uptake and trafficking of PLGA NPs and chitosan (Chi)-covered PLGA NPs in Madin-Darby bovine kidney (MDBK) and human colorectal adenocarcinoma (Colo 205) cells. Both PLGA and Chi-PLGA NPs were not cytotoxic to the studied cells at concentrations up to 2500 μg/mL. The positive charge conferred by the chitosan deposition on the PLGA NPs improved NPs uptake by MDBK cells. In this cell line, Chi-PLGA NPs colocalized partially with early endosomes compartment and showed a more consistent perinuclear localization than PLGA NPs. Kinetic uptake of PLGA NPs by Colo 205 was slower than that by MDBK cells, detected only at 24 h, exceeding that of Chi-PLGA NPs. This study offers new insights on NP interaction with target cells supporting the use of NPs as novel nutraceuticals/drug delivery systems in metabolic disorders or cancer therapy. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3599-3611, 2015.

  3. Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

    Science.gov (United States)

    Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

    2015-01-01

    Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth

  4. Synergistic effects of bovine respiratory syncytial virus and non-cytopathic bovine viral diarrhea virus infection on selected bovine alveolar macrophage functions.

    OpenAIRE

    Liu, L.; Lehmkuhl, H D; Kaeberle, M L

    1999-01-01

    The effect of bovine respiratory syncytial virus (BRSV) and non-cytopathic bovine viral diarrhea virus (ncpBVDV) infection on selected bovine alveolar macrophage (AM) functions was investigated. Alveolar macrophages were harvested from 2- to 6-month-old calves seronegative for BRSV and BVDV and inoculated with approximately 1 median cell culture infective dose of virus per AM. Control, BRSV infected, ncpBVDV-infected and BRSV-ncpBVDV coinfected AM cultures were evaluated for Fc receptor expre...

  5. Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

    Directory of Open Access Journals (Sweden)

    Cardoso Tereza C

    2012-05-01

    Full Text Available Abstract Background The possibility for isolating bovine mesenchymal multipotent cells (MSCs from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM. The three-dimensional (3D cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking. Results Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when

  6. Inhibition of JAK-STAT ERK/MAPK and Glycogen Synthase Kinase-3 Induces a Change in Gene Expression Profile of Bovine Induced Pluripotent Stem Cells

    Science.gov (United States)

    Malaver-Ortega, Luis F.; Sumer, Huseyin; Liu, Jun; Verma, Paul J.

    2016-01-01

    Pluripotent stem cells (PSCs) fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs) have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous REX1, OCT4, NANOG, and SOX2. Furthermore, LIF increased the levels of expression of OCT4 and REX1, compared with those cultured with LIF + bFGF. By contrast, bFGF decreased the levels of expression for both REX1 and OCT4. These results demonstrate that the biPSC gene expression profile is malleable by modification of the cell culture conditions well after nuclear reprogramming, and the culture conditions may determine their differentiation potential. PMID:26880968

  7. Inhibition of JAK-STAT ERK/MAPK and Glycogen Synthase Kinase-3 Induces a Change in Gene Expression Profile of Bovine Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Luis F. Malaver-Ortega

    2016-01-01

    Full Text Available Pluripotent stem cells (PSCs fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous REX1, OCT4, NANOG, and SOX2. Furthermore, LIF increased the levels of expression of OCT4 and REX1, compared with those cultured with LIF + bFGF. By contrast, bFGF decreased the levels of expression for both REX1 and OCT4. These results demonstrate that the biPSC gene expression profile is malleable by modification of the cell culture conditions well after nuclear reprogramming, and the culture conditions may determine their differentiation potential.

  8. Inhibition of JAK-STAT ERK/MAPK and Glycogen Synthase Kinase-3 Induces a Change in Gene Expression Profile of Bovine Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Malaver-Ortega, Luis F; Sumer, Huseyin; Liu, Jun; Verma, Paul J

    2016-01-01

    Pluripotent stem cells (PSCs) fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs) have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous REX1, OCT4, NANOG, and SOX2. Furthermore, LIF increased the levels of expression of OCT4 and REX1, compared with those cultured with LIF + bFGF. By contrast, bFGF decreased the levels of expression for both REX1 and OCT4. These results demonstrate that the biPSC gene expression profile is malleable by modification of the cell culture conditions well after nuclear reprogramming, and the culture conditions may determine their differentiation potential. PMID:26880968

  9. 25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner

    Directory of Open Access Journals (Sweden)

    Cantarutti Alyssa

    2010-11-01

    Full Text Available Abstract Background 25-hydroxycholesterol (25-OHC is a product of oxidation of dietary cholesterol present in human plasma. 25-OHC and other oxidized forms of cholesterol are implicated in modulating inflammatory responses involved in development of atherosclerosis and colon carcinogenesis. Methods Primary lymphatic, venous and arterial endothelial cells isolated from bovine mesentery (bmLEC, bmVEC, bmAEC were treated with 25-OHC and tested for several different cellular parameters. Results We found 25-OHC to be a potent inducer of cyclooxygenase-2 (Cox-2, prostaglandin G-H synthase-2 expression in bovine mesenteric lymphatic, venous, and arterial endothelial cells. The induction of Cox-2 expression in endothelial cells by 25-OHC led to an initial increase in cellular proliferation that was inhibited by the Cox-2 selective inhibitor celecoxib (Celebrex. Prolonged exposure to 25-OHC was cytotoxic. Furthermore, endothelial cells induced to express Cox-2 by 25-OHC were more sensitive to the effects of the Cox-2 selective inhibitor celecoxib (Celebrex. These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity. Conclusions Cox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient. Prolonged exposure to 25-OHC caused cell death and enhanced celecoxib-induced cell death in a cell-type dependent manner. The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

  10. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

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    Duban-Deweer Sophie

    2010-11-01

    Full Text Available Abstract Background Brain capillary endothelial cells (BCECs form the physiological basis of the blood-brain barrier (BBB. The barrier function is (at least in part due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein. Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets.

  11. Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways

    Science.gov (United States)

    Mills, I.; Cohen, C. R.; Kamal, K.; Li, G.; Shin, T.; Du, W.; Sumpio, B. E.

    1997-01-01

    Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these

  12. Regulation of pluripotency of inner cell mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2.

    Science.gov (United States)

    Dobbs, Kyle B; Khan, Firdous A; Sakatani, Miki; Moss, James I; Ozawa, Manabu; Ealy, Alan D; Hansen, Peter J

    2013-12-01

    Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9-16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.

  13. Effect of the ratios of unsaturated fatty acids on the expressions of genes related to fat and protein in the bovine mammary epithelial cells.

    Science.gov (United States)

    Sheng, R; Yan, S M; Qi, L Z; Zhao, Y L

    2015-04-01

    The objective of this study was to evaluate the effects of the different ratios of unsaturated fatty acids (UFAs) (oleic acid, linoleic acid, and linolenic acid) on the cell viability and triacylglycerol (TAG) content, as well as the mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Afterward, the cells were randomly allocated to six treatments, five UFA-treated groups, and one control group. For all of the treatments, the the fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L), and the cells were treated with different ratios of oleic, linoleic, and linolenic acids (0.75:4:1, 1.5:10:1, 2:13.3:1, 3:20:1, and 4:26.7:1) for 48 h, which were group 1 to group 5. The control culture solution contained only fatty acid-free BSA without UFAs (0 μM). The results indicated that the cell viability was not affected by adding different ratios of UFAs, but the accumulation of TAG was significantly influenced by supplementing with different ratios of UFAs. Adding different ratios of UFAs suppressed the expression of ACACA and FASN but had the opposite effect on the abundances of FABP3 and CD36 mRNA. The expression levels of PPARG, SPEBF1, CSN1S1, and CSN3 mRNA in the BMECs were affected significantly after adding different ratios of UFAs. Our results suggested that groups 1, 2, and 3 (0.75:4:1, 1.5:10:1, and 2:13.3:1) had stronger auxo-action on fat synthesis in the BMECs, where group 3 (2:13.3:1) was the best, followed by group 4 (3:20:1). However, group 5 (4:26.7:1) was the worst. Genes related to protein synthesis in the BMECs were better promoted in groups 2 and 3, and group 3 had the strongest auxo-action, whereas the present study only partly examined the regulation of protein synthesis at the transcriptional level; more studies on translation level are needed in the future

  14. Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1 blockade

    Directory of Open Access Journals (Sweden)

    Ikebuchi Ryoyo

    2011-09-01

    Full Text Available Abstract The inhibitory receptor programmed death-1 (PD-1 and its ligand, programmed death-ligand 1 (PD-L1 are involved in immune evasion mechanisms for several pathogens causing chronic infections. Blockade of the PD-1/PD-L1 pathway restores anti-virus immune responses, with concomitant reduction in viral load. In a previous report, we showed that, in bovine leukemia virus (BLV infection, the expression of bovine PD-1 is closely associated with disease progression. However, the functions of bovine PD-L1 are still unknown. To investigate the role of PD-L1 in BLV infection, we identified the bovine PD-L1 gene, and examined PD-L1 expression in BLV-infected cattle in comparison with uninfected cattle. The deduced amino acid sequence of bovine PD-L1 shows high homology to the human and mouse PD-L1. The proportion of PD-L1 positive cells, especially among B cells, was upregulated in cattle with the late stage of the disease compared to cattle at the aleukemic infection stage or uninfected cattle. The proportion of PD-L1 positive cells correlated positively with prediction markers for the progression of the disease such as leukocyte number, virus load and virus titer whilst on the contrary, it inversely correlated with the degree of interferon-gamma expression. Blockade of the PD-1/PD-L1 pathway in vitro by PD-L1-specific antibody upregulated the production of interleukin-2 and interferon-gamma, and correspondingly, downregulated the BLV provirus load and the proportion of BLV-gp51 expressing cells. These data suggest that PD-L1 induces immunoinhibition in disease progressed cattle during chronic BLV infection. Therefore, PD-L1 would be a potential target for developing immunotherapies against BLV infection.

  15. In vitro determination of carcinogenicity of sixty-four compounds using a bovine papillomavirus DNA-carrying C3H/10T(1/2) cell line.

    Science.gov (United States)

    Kowalski, L A; Laitinen, A M; Mortazavi-Asl, B; Wee, R K; Erb, H E; Assi, K P; Madden, Z

    2000-01-01

    A new in vitro test for predicting rodent carcinogenicity is evaluated against a testing database of 64 chemicals including both genotoxic and nongenotoxic carcinogens and carcinogens that normally require addition of an S-9 microsomal fraction for detection in the bacterial mutagenicity assay. The assay uses focus formation in a stable, bovine papillomavirus type 1 (BPV-1) DNA carrying C3H/10T(1/2) mouse embryo fibroblast cell line (T1) that does not require transfection, infection with virus, isolation of primary cells from animals, or addition of a microsomal fraction. Of a total database of 64 compounds, 92% of the carcinogens, promoters, or noncarcinogens were correctly predicted. Based on previously reported results, the test of bacterial mutagenicity would have correctly predicted 58% of carcinogens, promoters or noncarcinogens and the Syrian hamster embryo test would have correctly predicted 87% of carcinogens, promoters, or noncarcinogens of this database. Of carcinogens that normally require addition of an S-9 fraction, T1 cells correctly predicted rodent carcinogenicity of polyaromatic hydrocarbons, aflatoxins, azo-compounds, nitrosamines, and hydrazine without the addition of an S-9 fraction. Of nongenotoxic carcinogens, T1 cells correctly predicted diethylstilbestroel, diethylhexylphthalate, acetamides, alkyl halides, ethyl carbamate, and phorbol ester tumour promoters.

  16. A proteomic study of the differential protein expression in MDBK cells after bovine herpesvirus type 1 infection (BHV-1) strain treatment.

    Science.gov (United States)

    Guo, Li; Yang, Yanling; Liu, Linna; Liao, Peng; Wen, Yongjun; Wu, Hua; Cheng, Shipeng

    2015-01-01

    Different BHV-1 strains, such as the virulent IBRV LN01/08 strains and the attenuated vaccine strain IBRV LNM, produces different clinical immune responses; however, the study of the differential protein expression in Madin-Darby bovine kidney (MDBK) cells after BHV-1-infection still remains unclear. Here, we applied a comparative proteomic strategy, based on 2D and MALDI-TOF/MS platforms, to examine the differential expression of proteins in MDBK cells that were treated and not treated with virulent IBRV LN01/08 and attenuated IBRV LNM strains. A total of eight differential proteins, including pyruvate kinase, heat shock protein (HSP) 90 (HSP90AA1 and HSP90AB1), annexin A, albumin (ALB), scinderin (SCIN), tubulin (alpha 1a) and vimentin (VIM), were identified. Among these proteins, pyruvate kinase, and HSP90 (HSP90AB1), tubulin and vimentin were identified in the virulent IBRV LN01/08 strain group, but were not identified in the attenuated IBRV LNM group. These results play an important role in tumor formation and development, cell migration, tumor cell line apoptosis, cell invasion and viral infection. The HSP90 (HSP90AA1) protein was identified in the control group and the attenuated IBRV LNM-infected group. Most studies have shown that HSP90 proteins were more of a cancer gene target, and inhibiting its function would result to oncogene degradation during cancer treatment. On the other hand, ALB is associated to cell differentiation, apoptosis, necrosis, cell death, viral infection, autophagy, interstitial tissue inflammation, and cell survival. These results provide a theoretical basis for the systematic understanding of BHV-1-infection mechanisms and BHV-1-induced immune responses.

  17. Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus.

    OpenAIRE

    van Lierop, M J; Wagenaar, J P; Van Noort, J M; Hensen, E J

    1995-01-01

    Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.

  18. 78 FR 72979 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2013-12-04

    ... risks of other livestock diseases, such as bovine viral diarrhea, foot-and-mouth disease, infectious... Products Derived from Bovines,'' published in the Federal Register on September 18, 2007 (72 FR 53314-53379... 92, 93, 94, et al. Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine...

  19. Camel and bovine chymosin

    DEFF Research Database (Denmark)

    Jensen, Jesper Langholm; Mølgaard, Anne; Poulsen, Jens-Christian Navarro;

    2013-01-01

    Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite...... having 85% sequence identity, camel chymosin shows a 70% higher milk-clotting activity than bovine chymosin towards bovine milk. The activities, structures, thermal stabilities and glycosylation patterns of bovine and camel chymosin obtained by fermentation in Aspergillus niger have been examined...... differential scanning calorimetry revealed a slightly higher thermal stability of camel chymosin compared with bovine chymosin. The crystal structure of a doubly glycosylated variant of camel chymosin was determined at a resolution of 1.6 Å and the crystal structure of unglycosylated bovine chymosin...

  20. Cooperative cytotoxic activity of Zn and Cu in bovine serum albumin-conjugated ZnS/CuS nano-composites in PC12 cancer cells

    International Nuclear Information System (INIS)

    Series of self-assembled and mono-dispersed bovine serum albumin (BSA)-conjugated ZnS/CuS nano-composites with different Zn/Cu ratios had been successfully synthesized by a combination method of the biomimetic synthesis and ion-exchange strategy under the gentle conditions. High-resolution transmission electron microscopy observation, Fourier transform infrared spectra and zeta potential analysis demonstrated that BSA-conjugated ZnS/CuS nano-composites with well dispersity had the hierarchical structure and BSA was a key factor to control the morphology and surface electro-negativity of final products. The real-time monitoring by atomic absorption spectroscopy and powder X-ray diffraction revealed that the Zn/Cu ratio of nano-composites could be controlled by adjusting the ion-exchange time. In addition, the metabolic and morphological assays indicated that the metabolic proliferation and spread of rat pheochromocytoma (PC12) cells could be inhibited by nano-composites, with the high anti-cancer activity at a low concentration (4 ppm). What were more important, Zn and Cu in nano-composites exhibited a positive cooperativity at inhibiting cancer cell functions. The microscope observation and biochemical marker analysis clearly revealed that the nano-composites-included lipid peroxidation and disintegration of membrane led to the death of PC12 cells. Summarily, the present study substantiated the potential of BSA-conjugated ZnS/CuS nano-composites as anti-cancer drug

  1. Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer.

    Science.gov (United States)

    Wang, Y S; Zhao, X; Su, J M; An, Z X; Xiong, X R; Wang, L J; Liu, J; Quan, F S; Hua, S; Zhang, Y

    2011-03-01

    The objective of this study was to determine the effect of storage temperature during ovary transport on the developmental competence of bovine oocytes for use in somatic cell nuclear transfer (SCNT). Ovaries obtained from a slaughterhouse were stored in physiological saline for 3-4h at one of the three temperatures: 15 °C, 25 °C, or 35 °C. The developmental competence of oocytes used for SCNT was ascertained by cleavage and blastocyst formation rate, total cell number, apoptosis index, and the relative abundance of Bax and Hsp70.1 in day 7 blastocysts. Ovaries stored at 35 °C for 3-4h reduced the recovery rate of grade I and II oocytes compared with those stored at 25 °C or 15 °C (45.1±0.7% vs. 76.7±1.2% or 74.8±2.0%, Povaries stored at 15 °C, however, produced blastocysts with higher cell numbers (97.3±8.6 vs. 80.2±10.8 or 77.4±11.7; Povaries stored at 15 °C was lower than those stored at 25 °C or 35 °C (Pquality and developmental competence of oocytes used for SCNT due to the alleviation of stresses on the oocytes compared with those subjected to storage temperatures of 25 °C or 35 °C. PMID:21333472

  2. Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo.

    Science.gov (United States)

    Metzig, C; Grabowska, E; Eckert, K; Rehse, K; Maurer, H R

    1999-01-01

    The thiol protease, bromelain, an extract from pineapple stem, was suggested to have antithrombotic and anticoagulant activities in vivo. We studied the effects of bromelain on cell size distribution of isolated human platelets in vitro by Coulter Counter measurements. Preincubation of platelets with bromelain (10 micrograms/mL) completely prevented the thrombin (0.2 U/mL) induced platelet aggregation. Papain was less active in preventing platelet aggregation. In vitro, bromelain (0.1 microgram/mL) reduced the adhesion of bound, thrombin stimulated, fluorescent labeled platelets to bovine aorta endothelial cells. In addition, preincubation of platelets with bromelain, prior to thrombin, activation, reduced the platelet adhesion to the endothelial cells to the low binding value of unstimulated platelets. On the basis of mass concentrations, the proteases papain and trypsin were as effective as bromelain. Using a laser thrombosis model, the in vivo effects of orally and intraveneously applied bromelain on thrombus formation in rat mesenteric vessels were studied. Bromelain, orally applied at 60 mg/kg body weight, inhibited the thrombus formation in a time dependent manner, the maximum being after 2 hours in 11% of arterioles and 6% of venoles. Intravenous application at 30 mg/kg was slightly more active in reducing thrombus formation in arterioles (13%) and venoles (5%), suggesting that orally applied bromelain is biologically active. These results may help to explain some of the clinical effects observed after bromelain treatment in patients with thrombosis and related diseases.

  3. STIM1 positively regulates the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor in bovine aortic endothelial cells.

    Directory of Open Access Journals (Sweden)

    Éric Béliveau

    Full Text Available The endothelium is actively involved in many functions of the cardiovascular system, such as the modulation of arterial pressure and the maintenance of blood flow. These functions require a great versatility of the intracellular Ca2+ signaling that resides in the fact that different signals can be encoded by varying the frequency and the amplitude of the Ca2+ response. Cells use both extracellular and intracellular Ca2+ pools to modulate the intracellular Ca2+ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R, located on the endoplasmic reticulum (ER, is responsible for the release of Ca2+ from the intracellular store. The proteins STIM1 and STIM2 are also located on the ER and they are involved in the activation of a store-operated Ca2+ entry (SOCE. Due to their Ca2+ sensor property and their close proximity with IP3Rs on the ER, STIMs could modulate the activity of IP3R. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells and they both interact with IP3R. While STIM2 appears to play a minor role, STIM1 plays an important role in the regulation of agonist-induced Ca2+ mobilization in BAECs by a positive effect on both the SOCE and the IP3R-dependent Ca2+ release.

  4. Cooperative cytotoxic activity of Zn and Cu in bovine serum albumin-conjugated ZnS/CuS nano-composites in PC12 cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hua-Jie, E-mail: wanghuajie972001@163.com; Yu, Xue-Hong; Wang, Cai-Feng; Cao, Ying, E-mail: caoying1130@sina.com [Henan Normal University, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, College of Chemistry and Chemical Engineering (China)

    2013-11-15

    Series of self-assembled and mono-dispersed bovine serum albumin (BSA)-conjugated ZnS/CuS nano-composites with different Zn/Cu ratios had been successfully synthesized by a combination method of the biomimetic synthesis and ion-exchange strategy under the gentle conditions. High-resolution transmission electron microscopy observation, Fourier transform infrared spectra and zeta potential analysis demonstrated that BSA-conjugated ZnS/CuS nano-composites with well dispersity had the hierarchical structure and BSA was a key factor to control the morphology and surface electro-negativity of final products. The real-time monitoring by atomic absorption spectroscopy and powder X-ray diffraction revealed that the Zn/Cu ratio of nano-composites could be controlled by adjusting the ion-exchange time. In addition, the metabolic and morphological assays indicated that the metabolic proliferation and spread of rat pheochromocytoma (PC12) cells could be inhibited by nano-composites, with the high anti-cancer activity at a low concentration (4 ppm). What were more important, Zn and Cu in nano-composites exhibited a positive cooperativity at inhibiting cancer cell functions. The microscope observation and biochemical marker analysis clearly revealed that the nano-composites-included lipid peroxidation and disintegration of membrane led to the death of PC12 cells. Summarily, the present study substantiated the potential of BSA-conjugated ZnS/CuS nano-composites as anti-cancer drug.

  5. Graph clustering techniques applied to the glycomic response in glioblastoma cells to treatments with STAT3 phosphorylation inhibition and fetal bovine serum

    Science.gov (United States)

    Görke, Robert; Meyer-Bäse, Anke; Plant, Claudia; He, Huan; Emmett, Mark R.; Nilsson, Carol; Colman, Howard; Conrad, Charles A.

    2011-06-01

    Cancer stem cells (CSC) represent a very small percentage of the total tumor population however they pose a big challenge in treating cancer. Glycans play a key role in cancer therapeutics since overexpression of them depending on the glycan type can lead either to cell death or more invasive metastasis. Two major components, fetal bovine serum (FBS) and STAT3, are known to up- or down-regulate certain glycolipid or phospholipid compositions found in glioblastoma CSCs. The analysis and the understanding of the global interactional behavior of lipidomic networks remains a challenging task and can not be accomplished solely based on intuitive reasoning. The present contribution aims at applying graph clustering networks to analyze the functional aspects of certain activators or inhibitors at the molecular level in glioblastoma stem cells (GSCs). This research enhances our understanding of the differences in phenotype changes and determining the responses of glycans to certain treatments for the aggressive GSCs, and represents together with a quantitative phosphoproteomic study1 the most detailed systems biology study of GSCs differentiation known so far. Thus, these new paradigms are providing unique understanding of the mechanisms involved in GSC maintenance and tumorigenicity and are thus opening a new window to biomedical frontiers.

  6. Exploring the membrane topology of prohormone convertase 1 in AtT20 Cells: in situ analysis by immunofluorescence microscopy [v1; ref status: indexed, http://f1000r.es/QFfyFd

    Directory of Open Access Journals (Sweden)

    Niamh X Cawley

    2012-08-01

    Full Text Available Prohormone convertase 1 (PC1 was previously characterized as a partially transmembrane protein in purified chromaffin granules of bovine adrenal medulla1. This was challenged with experiments on transfected PC1 in COS1 cells, a non-endocrine cell line2. To address this issue, we undertook to analyze its extraction properties in vitro and its immunocytochemical localization in situ in AtT20 cells, an endocrine cell line that expresses PC1. Most of the 87 kDa form of PC1 was resistant to carbonate extraction suggesting that it had properties of a transmembrane protein. Under semi-permeabilized conditions whereby only the plasma membrane was permeabilized, the carboxy-terminus of PC1 was specifically immunostained whereas the amino-terminus was not. These results indicate that the amino-terminus of PC1 was within the lumen of the Golgi and granules, and some of the C-terminus was exposed to the cytosol. Thus, endogenous PC1 can assume a transmembrane orientation in situ in AtT20 cells.

  7. 78 FR 73993 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2013-12-10

    ... Health Inspection Service 9 CFR Parts 92, 93, 94, 95, 96, and 98 RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products Corrections In rule document 2013-28228 appearing...

  8. 77 FR 20319 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-04-04

    ...; ] DEPARTMENT OF AGRICULTURE Animal and Plant Health Inspection Service 9 CFR Part 93 RIN 0579-AC68 Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products Correction In proposed rule...

  9. Polymorphisms in bovine immune genes and their associations with somatic cell count and milk production in dairy cattle

    Directory of Open Access Journals (Sweden)

    Magee David A

    2010-11-01

    Full Text Available Abstract Background Mastitis, an inflammation of the mammary gland, is a major source of economic loss on dairy farms. The aim of this study was to quantify the associations between two previously identified polymorphisms in the bovine toll-like receptor 2 (TLR2 and chemokine receptor 1 (CXCR1 genes and mammary health indictor traits in (a 246 lactating dairy cow contemporaries representing five breeds from one research farm and (b 848 Holstein-Friesian bulls that represent a large proportion of the Irish dairy germplasm. To expand the study, a further 14 polymorphisms in immune genes were included for association studies in the bull population. Results TLR4-2021 associated (P SERPINA1 haplotype with superior genetic merit for milk protein yield and milk fat percentage (P Conclusion Of the sixteen polymorphisms in seven immune genes genotyped, just CXCR1-777 tended to associate with SCS, albeit only in the on-farm study. The lack of an association between the polymorphisms with SCS in the Holstein-Friesian data set would question the potential importance of these variants in selection for improved mastitis resistance in the Holstein-Friesian cow.

  10. Antimicrobial and cell viability measurement of bovine serum albumin capped silver nanoparticles (Ag/BSA) loaded collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film.

    Science.gov (United States)

    Bakare, Rotimi; Hawthrone, Samantha; Vails, Carmen; Gugssa, Ayele; Karim, Alamgir; Stubbs, John; Raghavan, Dharmaraj

    2016-03-01

    Bacterial infection of orthopedic devices has been a major concern in joint replacement procedures. Therefore, this study is aimed at formulating collagen immobilized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film loaded with bovine serum albumin capped silver nanoparticles (Ag/BSA NPs) to inhibit bacterial growth while retaining/promoting osteoblast cells viability. The nanoparticles loaded collagen immobilized PHBV film was characterized for its composition by X-ray Photoelectron Spectroscopy and Anodic Stripping Voltammetry. The extent of loading of Ag/BSA NPs on collagen immobilized PHBV film was found to depend on the chemistry of the functionalized PHBV film and the concentration of Ag/BSA NPs solution used for loading nanoparticles. Our results showed that more Ag/BSA NPs were loaded on higher molecular weight collagen immobilized PHEMA-g-PHBV film. Maximum loading of Ag/BSA NPs on collagen immobilized PHBV film was observed when 16ppm solution was used for adsorption studies. Colony forming unit and optical density measurements showed broad antimicrobial activity towards Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa at significantly lower concentration i.e., 0.19 and 0.31μg/disc, compared to gentamicin and sulfamethoxazole trimethoprim while MTT assay showed that released nanoparticles from Ag/BSA NPs loaded collagen immobilized PHBV film has no impact on MCTC3-E1 cells viability.

  11. Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells.

    Science.gov (United States)

    Jarmołowska, Beata; Teodorowicz, Małgorzata; Fiedorowicz, Ewa; Sienkiewicz-Szłapka, Edyta; Matysiewicz, Michał; Kostyra, Elżbieta

    2013-11-01

    Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca(2+) concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.

  12. N-doped carbon dots derived from bovine serum albumin and formic acid with one- and two-photon fluorescence for live cell nuclear imaging.

    Science.gov (United States)

    Tan, Mingqian; Li, Xintong; Wu, Hao; Wang, Beibei; Wu, Jing

    2015-12-01

    Carbon dots with both one- and two-photon fluorescence have drawn great attention for biomedical imaging. Herein, nitrogen-doped carbon dots were facilely developed by one-pot hydrothermal method using bovine serum albumin and formic acid as carbon sources. They are highly water-soluble with strong fluorescence when excited with ultraviolet or near infrared light. The carbon dots have a diameter of ~8.32 nm and can emit strong two-photon induced fluorescence upon excitation at 750 nm with a femtosecond laser. X-ray photoelectron spectrometer analysis revealed that the carbon dots contained three components, C, N and O, corresponding to the peak at 285, 398 and 532 eV, respectively. The Fourier-transform infrared spectroscopy analysis revealed that there are carboxyl and carboxylic groups on the surface, which allowed further linking of functional molecules. pH stability study demonstrated that the carbon dots are able to be used in a wide range of pH values. The fluorescence mechanism is also discussed in this study. Importantly, these carbon dots are biocompatible and highly photostable, which can be directly applied for both one- and two-photon living cell imaging. After proper surface functionalization with TAT peptide, they can be used as fluorescent probes for live cell nuclear-targeted imaging.

  13. Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon-γ-induced malignant transformation of primary bovine mammary epithelial cells

    Science.gov (United States)

    Xia, X-j; Gao, Y-y; Zhang, J; Wang, L; Zhao, S; Che, Y-y; Ao, C-j; Yang, H-j; Wang, J-q; Lei, L-c

    2016-01-01

    Autophagy has been linked to the regulation of both the prevention and progression of cancer. IFN-γ has been shown to induce autophagy in multiple cell lines in vitro. However, whether IFN-γ can induce autophagy and whether autophagy promotes malignant transformation in healthy lactating bovine mammary epithelial cells (BMECs) remain unclear. Here, we provide the first evidence of the correlation between IFN-γ treatment, autophagy and malignant transformation and of the mechanism underlying IFN-γ-induced autophagy and subsequent malignant transformation in primary BMECs. IFN-γ levels were significantly increased in cattle that received normal long-term dietary corn straw (CS) roughage supplementation. In addition, an increase in autophagy was clearly observed in the BMECs from the mammary tissue of cows expressing high levels of IFN-γ. In vitro, autophagy was clearly induced in primary BMECs by IFN-γ within 24 h. This induced autophagy could subsequently promote dramatic primary BMEC transformation. Furthermore, we found that IFN-γ promoted arginine depletion, activated the general control nonderepressible-2 kinase (GCN2) signalling pathway and resulted in an increase in autophagic flux and the amount of autophagy in BMECs. Overall, our findings are the first to demonstrate that arginine depletion and kinase GCN2 expression mediate IFN-γ-induced autophagy that may promote malignant progression and that immunometabolism, autophagy and cancer are strongly correlated. These results suggest new directions and paths for preventing and treating breast cancer in relation to diet. PMID:27551491

  14. Unlocking the bovine genome

    Science.gov (United States)

    The draft genome sequence of cattle (Bos taurus) has now been analyzed by the Bovine Genome Sequencing and Analysis Consortium and the Bovine HapMap Consortium, which together represent an extensive collaboration involving more than 300 scientists from 25 different countries. ...

  15. Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

    Science.gov (United States)

    Bevacqua, R J; Fernandez-Martín, R; Savy, V; Canel, N G; Gismondi, M I; Kues, W A; Carlson, D F; Fahrenkrug, S C; Niemann, H; Taboga, O A; Ferraris, S; Salamone, D F

    2016-11-01

    The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P genetic modifications (29) was higher than the total number of gene-edited embryos, as

  16. Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

    Science.gov (United States)

    Bevacqua, R J; Fernandez-Martín, R; Savy, V; Canel, N G; Gismondi, M I; Kues, W A; Carlson, D F; Fahrenkrug, S C; Niemann, H; Taboga, O A; Ferraris, S; Salamone, D F

    2016-11-01

    The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was

  17. Survey on vertical infection of bovine viral diarrhea virus from fetal bovine sera in the field

    OpenAIRE

    NAGAYAMA, Kumiko; OGUMA, Keisuke; SENTSUI, Hiroshi

    2015-01-01

    Bovine viral diarrhea virus (BVDV) isolation and antibody survey were performed using 2,758 fetal bovine sera (FBS) collected from slaughterhouses in New Zealand, Australia and the Dominican Republic, and then sent to Japan to manufacture commercial serum for cell culture use. FBS in the Dominican Republic were pooled for each several individuals, and those collected in other countries were separated according to each individual and subjected to the tests. BVDV was isolated from 25 (0.91%) FB...

  18. In vitro protective effect of bacteria-derived bovine alpha interferon I1 against selected bovine viruses.

    OpenAIRE

    Gillespie, J H; Robson, D. S.; Scott, F. W.; Schiff, E I

    1985-01-01

    We used bacteria-derived bovine alpha-interferon I1 (Bo IFN-alpha I1) to study its antiviral effect in a bovine turbinate cell line on bovine diarrhea virus, infectious bovine rhinotracheitis virus, parainfluenza 3 virus, and pseudorabies virus. We based our study upon replicate tests for each strain by using a block titration system with various concentrations of Bo IFN-alpha I1 against various concentrations of virus. The data were compiled in two-axis tables (replicate X concentration) and...

  19. Characterisation of bovine epiblast-derived outgrowth colonies

    DEFF Research Database (Denmark)

    Østrup, Esben; Gjørret, Jakob; Schauser, Kirsten Hallundbæk;

    2010-01-01

    The aim of the present study was to characterise bovine epiblast-derived outgrowth colonies (OCs) with respect to the embryonic origin of their cellular components. Epiblasts were isolated mechanically from bovine Day 12 embryos. Epiblasts were cultured on feeder layers of SNL cells (neomycin...

  20. Changes in some pro-and anti-inflammatory cytokines produced by bovine peripheral blood mononuclear cells following foot and mouth disease vaccination

    Directory of Open Access Journals (Sweden)

    N. Delirezh

    2016-09-01

    Full Text Available Interleukin (IL-17 is exclusively produced by CD4 helper T-cells upon activation. It most often acts as a pro-inflammatory cytokine, which stimulates the release of pro-inflammatory cytokines IL-6, IL-8, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF. In this study, we studied the in-vitro IL-17 response to specific antigens and a variety of mitogens and compared the IL-17 response to IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-γ responses. We used a foot and mouth disease (FMD vaccine as specific antigens and mitogens (phytohemagglutinin [PHA], pokeweed mitogen [PWM], and concanavalin A [Con A] to stimulate peripheral blood mononuclear cells (PBMCs of vaccinated calves. Cell culture supernatant was harvested and analyzed for cytokines, using commercially available bovine ELISA kits. The mitogens induced a significant increase in IL-17 production. IL-17 was produced at high levels in response to the T cell-stimulated mitogens, PHA, and Con A, and at low levels in response to PWM mitogens. In contrast, level of the produced IL-17 cytokines in response to the FMDV antigens was lower as compared to those produced by mitogens. The FMDV antigens and mitogens significantly increased IL-17 production. There was not a correlation between IL-17 production and type-1 cytokine, IFN-γ, and IL-2, while there was a correlation between type-2 cytokine, IL-4, and IL-5 at either cytokine level produced by PBMCs stimulated by FMDV antigens. Moreover, there was an interaction between IL-17 and IL-6, that is, as IL-6 cytokine level elevated or diminished, IL-17 cytokine level increased or decreased, as well.

  1. Evaluation of anorganic bovine-derived hydroxyapatite matrix/cell binding peptide as a bone graft material in the treatment of human periodontal infrabony defects: A clinico-radiographic study

    OpenAIRE

    Ghousia Fatima; Ravindra Shivamurthy; Srinath Thakur; Mohammad Abdul Baseer

    2015-01-01

    Background: Various bone graft materials have been used in the treatment of periodontal defects. A synthetic bone substitute material composed of P-15 with anorganic bone mineral has been scantly studied. Hence, the present study was aimed to evaluate and compare the efficacy of anorganic bovine-derived hydroxyapatite matrix (ABM)/cell binding peptide (P-15) in human periodontal infrabony defects with that of open flap debridement (OFD) alone. Materials and Methods: A split-mouth, randomized ...

  2. Antibacterial activity and immunomodulatory effects on a bovine mammary epithelial cell line exerted by nisin A-producing Lactococcus lactis strains.

    Science.gov (United States)

    Malvisi, M; Stuknytė, M; Magro, G; Minozzi, G; Giardini, A; De Noni, I; Piccinini, R

    2016-03-01

    Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl-β-d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor-α downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor-α release could be an interesting approach to reduce the symptoms linked

  3. Bovine Herpesvirus 4 infections and bovine mastitis

    NARCIS (Netherlands)

    Wellenberg, Gerardus Johannus

    2002-01-01

    Mastitis is an often occurring disease in dairy cattle with an enormous economic impact for milk producers worldwide. Despite intensive research, which is historically based on the detection of bacterial udder pathogens, still around 20-35% of clinical cases of bovine mastitis have an unknown aetiol

  4. Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells.

    Science.gov (United States)

    Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M; Rice, Kenner C; Gannon, Brenda M; Fantegrossi, William E; Gonzalez, Carmen; Paule, Merle G; Ali, Syed F

    2016-08-26

    Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic. PMID:27320055

  5. In vitro exposure of Penicillium mycotoxins with or without a modified yeast cell wall extract (mYCW) on bovine macrophages (BoMacs).

    Science.gov (United States)

    Oh, Se-Young; Quinton, V Margaret; Boermans, Herman J; Swamy, H V L N; Karrow, Niel A

    2015-11-01

    Penicillium mycotoxins (PMs) are contaminants that are frequently found in grain or crop-based silage for animal feed. Previously, we have characterized the potential immunotoxicity of the following PMs: citrinin (CIT), ochratoxin A (OTA), patulin (PAT), mycophenolic acid (MPA), and penicillic acid (PA) by using a bovine macrophage cell line (BoMacs). In the present study, cell proliferation was used as a bioassay endpoint to evaluate the efficacy of a modified yeast cell wall extract (mYCW), for preventing PM toxicity under various in vitro conditions such as the following: pH (3, 5, 7), incubation time (1, 2, 4, 6 h), percentage of mYCW (0.05, 0.1, 0.2, 0.5, 1.0 %), and PM concentration. mYCW was most effective in preventing the toxicity of 12.88 and 25.8 μM OTA at pH 3.0 (p < 0.0001), regardless of incubation time (p < 0.0001) and the percentage of mYCW (p < 0.0001). An incubation time of 6 h (p < 0.05) or 0.5 and 1.0 % mYCW (p < 0.0001) significantly improved the efficacy of mYCW for preventing CIT toxicity. In contrast, 0.5 and 1.0 % of mYCW appeared to exacerbate the PAT toxicity (p < 0. 0001). This effect on PAT toxicity was constantly observed with higher PAT concentrations, and it reached significance at a concentration of 0.70 μM (p < 0.0001). mYCW had no effect on PA toxicity. These results suggest that mYCW may reduce OTA toxicity and, to some extent, CIT toxicity at pH 3.0. Although PAT toxicity was increased by mYCW treatment, PAT is readily degraded during heat treatment and may therefore be dealt with using other preventative measures.

  6. Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts.

    Science.gov (United States)

    Zuniga, Eveline; Melville, Priscilla A; Saidenberg, André B S; Laes, Marco A; Gonsales, Fernanda F; Salaberry, Sandra R S; Gregori, Fabio; Brandão, Paulo E; dos Santos, Franklin G B; Lincopan, Nilton E; Benites, Nilson R

    2015-12-01

    This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process.

  7. Genetic characterization of bovine viral diarrhea virus strains in Beijing, China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle.

    Science.gov (United States)

    Weng, Xiao Gang; Song, Quan Jiang; Wu, Qiong; Liu, Ming Chao; Wang, Meng Ling; Wang, Jiu Feng

    2015-01-01

    To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5'-untranslated region (5'-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.

  8. Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts.

    Science.gov (United States)

    Zuniga, Eveline; Melville, Priscilla A; Saidenberg, André B S; Laes, Marco A; Gonsales, Fernanda F; Salaberry, Sandra R S; Gregori, Fabio; Brandão, Paulo E; dos Santos, Franklin G B; Lincopan, Nilton E; Benites, Nilson R

    2015-12-01

    This study aimed to elucidate aspects of the epidemiology of bovine subclinical mastitis through the assessment of genes encoding MSCRAMM (microbial surface components recognizing adhesive matrix molecules - a group of adhesins) and protein Bap (implicated in biofilm formation), in coagulase-positive (CPS) and coagulase-negative (CNS) Staphylococcus isolated from subclinical mastitis. Milk samples were collected for microbiological exams, somatic cell count (SCC) and a survey of the genes coding for MSCRAMM (cna, eno, ebpS, fnbA, fnbB and fib) and biofilm-associated protein Bap (bap) in 106 Staphylococcus spp. isolates using PCR. The frequencies of occurrence of eno (82.1%), fnbA (72.6%), fib (71.7%) and bap (56.6%) were higher (P < 0.0001) compared with the other assessed genes (cna, ebpS and fnbB). The higher frequency of occurrence (P < 0.005) of the bap gene in CNS compared with CPS suggests that in these species biofilm formation is an important mechanism for the persistence of the infection. The medians of the SCCs in the samples where eno, fnbA, fib and bap genes were detected were higher compared with Staphylococcus without the assessed genes (P < 0.05) and negative samples (P < 0.01), which indicated that the presence of these MSCRAMM may be related to a higher intensity of the inflammatory process. PMID:26318876

  9. Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

    Science.gov (United States)

    van Tol, H T; van Eijk, M J; Mummery, C L; van den Hurk, R; Bevers, M M

    1996-10-01

    Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles. PMID:8914080

  10. Expression of a 50 kDa putative receptor for bovine viral diarrhea virus in bovine fetal tissues.

    OpenAIRE

    Zheng, L; Zhang, S.; W. Xue; Kapil, S; Minocha, H C

    1998-01-01

    The expression of a 50 kDa bovine viral diarrhea virus putative receptor in different bovine fetal tissues from 3-month old fetuses was studied. The receptor expression was examined by immunocytochemical staining and by immunoblotting using antiidiotypic probe (anti-D89). Intense specific staining in enterocytes of the small and large intestines, cortical tubular epithelial cells of kidneys, respiratory epithelial cells of the trachea and esophageal mucosal epithelial cells was observed, demo...

  11. Biphasic activation of PI3K/Akt and MAPK/Erk1/2 signaling pathways in bovine herpesvirus type 1 infection of MDBK cells

    Directory of Open Access Journals (Sweden)

    Zhu Liqian

    2011-04-01

    Full Text Available Abstract Many viruses have been known to control key cellular signaling pathways to facilitate the virus infection. The possible involvement of signaling pathways in bovine herpesvirus type 1 (BoHV-1 infection is unknown. This study indicated that infection of MDBK cells with BoHV-1 induced an early-stage transient and a late-stage sustained activation of both phosphatidylinositol 3-kinase (PI3K/Akt and mitogen activated protein kinases/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2 signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective inhibitor, Ly294002 for PI3K and U0126 for MAPK kinase (MEK1/2, respectively. Furthermore, treatment of MDBK cells with Ly294002 caused a 1.5-log reduction in virus titer, while U0126 had little effect on the virus production. In addition, the inhibition effect of Ly294002 mainly occurred at the post-entry stage of the virus replication cycle. This revealed for the first time that BoHV-1 actively induced both PI3K/Akt and MAPK/Erk1/2 signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.

  12. Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways

    Science.gov (United States)

    Mills, I.; Cohen, C. R.; Kamal, K.; Li, G.; Shin, T.; Du, W.; Sumpio, B. E.

    1997-01-01

    Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 microM Rp-cAMP) or PKC (with 5-20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses.

  13. Non-classical effects of prolactin on the innate immune response of bovine mammary epithelial cells: Implications during Staphylococcus aureus internalization.

    Science.gov (United States)

    Medina-Estrada, Ivan; Alva-Murillo, Nayeli; López-Meza, Joel E; Ochoa-Zarzosa, Alejandra

    2015-12-01

    Staphylococcus aureus has the ability to invade mammary epithelial cells (bMECs) causing mastitis. This event depends primarily on the α5β1 integrin in the host cell. In addition, bMECs are a target for the hormone prolactin (PRL), which can regulate β1 integrin-dependent actions related to differentiation and lactation. Previously, we demonstrated that bovine PRL (bPRL, 5 ng/ml) stimulates S. aureus internalization into bMECs. TLR2 is important during S. aureus infections, but its activation by PRL has not yet been established. The objective of this study was to determine the role of α5β1 integrin and TLR2 during S. aureus internalization into bMECs stimulated with bPRL. We demonstrated that the prolactin-stimulated internalization of S. aureus decreases in response to the blockage of α5β1 integrin (∼ 80%) and TLR2 (∼ 80%). bPRL increases the membrane abundance (MA) of α5β1 integrin (∼ 20%) and induces TLR2 MA (∼ 2-fold). S. aureus reduces the α5β1 integrin MA in bMECs treated with bPRL (∼ 75%) but induces TLR2 MA in bMECs (∼ 3-fold). Bacteria and bPRL did not modify TLR2 MA compared with the hormone alone. S. aureus induces the activation of the transcription factor AP-1, which was inhibited in bMECs treated with bPRL and infected. In general, bPRL induces both pro- and anti-inflammatory responses in bMECs, which are abated in response to bacterial challenge. Interestingly, the canonical Stat-5 transcription factor was not activated in the challenged bMECs and/or treated with bPRL. Taken together, these results support novel functions of prolactin as a modulator of the innate immune response that do not involve the classical prolactin pathway.

  14. The effect of DDT and its metabolite (DDE) on prostaglandin secretion from epithelial cells and on contractions of the smooth muscle of the bovine oviduct in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wrobel, Michal H.; Mlynarczuk, Jaroslaw; Kotwica, Jan, E-mail: janko@pan.olsztyn.pl

    2012-03-01

    The insecticide DDT and its metabolite (DDE), due to their lipolytic nature and resistance to biodegradation, are accumulated in the living tissues. In cows, DDT and DDE were found to affect prostaglandin (PG) secretion from the endometrium and contractions of the myometrium. In this study, the impact of both xenobiotics (0.1, 1, 10 or 100 ng/ml) on the function of epithelial cells and muscle strips of bovine oviducts from 1 to 5 day of the oestrous cycle was examined. Therefore the concentration of PGE2 and PGFM (a metabolite of PGF2α) in culture media, mRNA expression of genes involved in PGs synthesis in epithelial cells and the force and amplitude of strips contractions were measured after 2 and 24 or 48 h of incubation. Neither DDT nor DDE affected the viability of cells after 48 h (P > 0.05). Both DDT and DDE increased the concentrations of PGFM in culture medium and secretion of PGE2 after only 2 h of cell culture (P < 0.05). Similar effects were seen for the influence of DDE on amount of PGFM after 48 h, while DDT decreased secretion of PGE2 (P < 0.05). DDT after 2 h increased (P < 0.05) mRNA expression of PGF2α synthase (PGFS), while both xenobiotics decreased (P < 0.05) mRNA expression of cyclooxygenase-2 (COX-2) after 24 h. DTT also increased the force of isthmus contractions after 2 h, as did both xenobiotics after 48 h (P < 0.05). Moreover, after 2 and 48 h, DDE stimulated the amplitude of contractions of the isthmus as well as the ampulla, (P < 0.05). The effect of both compounds on oviduct contractions was diminished by indomethacin, which blocks PG synthesis. We conclude that oviductal secretion of prostaglandins is affected, by DDT and DDE. The influence of these xenobiotics on PGF2α and PGE2 secretion and ratio may be part of the mechanism by which both DDT and its metabolite disturb the contractions of oviductal muscle. -- Highlights: ► DDT and its metabolite – DDE are accumulated in the living tissues. ► The insecticides affected PGF2

  15. Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles.

    Science.gov (United States)

    Farini, Veronica L; Camaño, Carla V; Ybarra, Gabriel; Viale, Diego L; Vichera, Gabriel; Yakisich, Juan S; Radrizzani, Martín

    2016-07-10

    In cattle, cryopreservation of semen and sex-sorting kill up to 50% of spermatozoa and decrease the success of assisted insemination (AI). Therefore, significant efforts are being carried out to improve the quality of semen prior to AI. In this work we used the Cell-SELEX technique to select single strand DNA aptamers able to recognize with high affinity and specificity damaged sperm cells generated by heat-treatment. We first isolated aptamers with a conserved two motifs of 6 nucleotides of length that bind to the membrane of heat-treated spermatozoa. Then, we used synthetic biotin-labeled aptamers containing the conserved motif to recognize membrane-damaged cells and separate them from viable cells by the use of avidin-coated superparamagnetic iron oxide nanoparticles (SPION). This procedure improved the quality of semen by significantly increasing the percentage of healthy sperm cells without affecting the rate of blastocyst cleavage. This technique was successfully applied to both unsorted and sex-sorted sperm suspension. PMID:27164256

  16. Short communication: the pharmacological peroxisome proliferator-activated receptor α agonist WY-14,643 increases expression of novel organic cation transporter 2 and carnitine uptake in bovine kidney cells.

    Science.gov (United States)

    Zhou, X; Wen, G; Ringseis, R; Eder, K

    2014-01-01

    Recent studies in rodents demonstrated that peroxisome proliferator-activated receptor α (PPARα), a central regulator of energy homeostasis, is an important transcriptional regulator of the gene encoding the carnitine transporter novel organic cation transporter 2 (OCTN2). Less is known with regard to the regulation of OCTN2 by PPARα and its role for carnitine transport in cattle, even though PPARα activation physiologically occurs in the liver of high-producing cows during early lactation. To explore the role of PPARα for OCTN2 expression and carnitine transport in cattle, we studied the effect of the PPARα activator WY-14,643 on the expression of OCTN2 in the presence and absence of PPARα antagonists and on OCTN2-mediated carnitine transport in the Madin-Darby bovine kidney (MDBK) cell line. The results show that WY-14,643 increases mRNA and protein levels of OCTN2, whereas co-treatment of MDBK cells with WY-14,643 and the PPARα antagonist GW6471 blocks the WY-14,643-induced increase in mRNA and protein levels of OCTN2 in bovine cells. In addition, treatment of MDBK cells with WY-14,643 stimulates specifically Na(+)-dependent carnitine uptake in MDBK cells, which is likely the consequence of the increased carnitine transport capacity of cells due to the elevated expression of OCTN2. In conclusion, our results indicate that OCTN2 expression and carnitine transport in cattle, as in rodents, are regulated by PPARα.

  17. A prebiotic, Celmanax™, decreases Escherichia coli O157:H7 colonization of bovine cells and feed-associated cytotoxicity in vitro

    Directory of Open Access Journals (Sweden)

    Juba Jean

    2011-04-01

    Full Text Available Abstract Background Escherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax™, or probiotic, Dairyman's Choice™ paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases. Findings Dairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillusflavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs

  18. Remodeling of bovine endometrium throughout the estrous cycle.

    Science.gov (United States)

    Arai, Miki; Yoshioka, Shin; Tasaki, Yukari; Okuda, Kiyoshi

    2013-11-01

    The mammalian endometrium changes morphologically and functionally throughout the estrous cycle. In some species, endometrial cells also undergo periodic proliferation and degeneration. However, the remodeling of bovine endometrium throughout the estrous cycle remains unclear. In the present study, we examined how the remodeling of bovine endometrium varied through the estrous cycle by measuring the relative rates of cell proliferation and apoptosis. Cells positive for both KI-67 (a proliferation marker) and cleaved caspase-3 (CCP3: an apoptotic cell marker) were immunohistochemically evaluated throughout the estrous cycle in the luminal and glandular epithelia, and the stroma of bovine endometrium. Percentages of KI-67-positive cells tended to be higher at the early luteal and follicular stages than at the mid and late luteal stages in all cell types. Similarly, percentages of CCP3-positive cells were higher at the early luteal stage than at the mid and late luteal stages in the luminal epithelium and stroma. Furthermore, CCP3 expression levels by Western blot analysis agreed with these immunohistological observations. On the other hand, DNA fragmentation was detected in the bovine endometrium without significant differences during the estrous cycle by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Together, these results show that cell proliferation and apoptosis undergo cyclic patterns in the bovine endometrium, and suggest that the bovine endometrium is remodeled in each estrous cycle. PMID:24051170

  19. Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods

    DEFF Research Database (Denmark)

    van der Valk, J; Brunner, D; De Smet, K;

    2010-01-01

    , reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement...... with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult...

  20. Adipogenesis of bovine perimuscular preadipocytes

    International Nuclear Information System (INIS)

    In this study, non-transformed progeny adipofibroblasts, derived from mature adipocyte dedifferentiation, was used as a novel in vitro model to study adipogenic gene expression in cattle. Adipofibroblasts from dedifferentiated mature perimuscular fat (PMF) tissue were cultured with differentiation stimulants until the cells exhibited morphological differentiation. Treated cells were harvested from day 2 to 16 for RNA extraction, whereas control cells were cultured without addition of stimulants. Results from time course gene expression assays by quantitative real-time PCR revealed that peroxisome proliferator-activated receptor gamma (PPAR-γ), sterol regulatory element binding protein 1 (SREBP-1) and their six down-stream genes were co-expressed at day 2 post-differentiation induction. When compared to other adipogenesis culture systems, the adipogenic gene expression of bovine PMF adipofibroblasts culture was different, especially to the rodent model. Collectively, these results demonstrated PPAR-γ and SREBP-1 cooperatively play a key role to regulate the re-differentiation of bovine adipofibroblasts, during early conversion stages in vitro

  1. MicroRNA (miRNA) expression is regulated by butyrate induced epigenetic modulation of gene expression in bovine cells

    Science.gov (United States)

    We present evidence that butyrate induced histone acetylation regulates miRNA expression. MicroRNA expression microarray profiling revealed that 35 miRNA transcripts are significantly (p cells were treated with 10 mM butyrate. Among them, 11 transcripts are dif...

  2. ChIp-seq of bovine cells (MDBK) to study butyrate-induced histone modification with 10 datasets

    Science.gov (United States)

    Next-generation sequencing was combined with chromatin immunoprecipitation (ChIP) technology to analyze histone modification (acetylation) induced by butyrate and to map the epigenomic landscape of normal histone H3, H4 in rumen cells of the cow. Ten variants of histone H3 and H4 modification were m...

  3. 不同新生牛血清对体外培养BHK-21细胞的影响%Effect of various types of newborn bovine sera on culture in vitro of BHK-21 cells

    Institute of Scientific and Technical Information of China (English)

    孙招金; 陈柄企; 王玲; 叶贺佳; 童菊芸; 罗开健

    2012-01-01

    目的 比较不同新生牛血清对体外培养的BHK-21细胞的影响.方法 以不同厂家和同一厂家不同批次的共6种新生牛血清培养BHK-21细胞,观察细胞传代后在不同血清培养条件下的形态、增长倍数和细胞维持时间.结果 6号血清培养的细胞生长状态最好,增长倍数最高,维持细胞存活时间最长;3号血清培养效果次之;2号、4号和5号血清培养的细胞至第4天仍未长满单层;1号血清培养的细胞基本未生长.结论 不同厂家和同一厂家不同批次的新生牛血清对BHK-21细胞体外培养的效果存在差异.%Objective To compare the effect of various types of newborn bovine sera on culture in vitro of BHK-21 cells. Methods BHK-21 cells were cultured with six kinds of newborn bovine sera,manufactured by various manufacturers or of different batches manufactured by the same manufacturer,respectively,and observed for morphology,increased fold in quantity,and survival time after subculture. Results The cells cultured with serum No. 6 grew well,of which the increased fold in quantity was high and survival time was long as compared with those cultured with other newborn bovine sera. The culture efficacy of cells with serum No. 3 was only second to that with serum No.6. However,no monolayer was formed in the cells on day 4 after culture with sera No. 2,4 and 5,and little growth was observed in the cells cultured with serum No.l. Conclusion Significant differences were observed in culture efficacies of BHK-21 cells with newborn bovine sera manufactured by various manufacturers or of different batches manufactured by the same manufacturer.

  4. Deep Sequencing and Screening of Differentially Expressed MicroRNAs Related to Milk Fat Metabolism in Bovine Primary Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Binglei Shen

    2016-02-01

    Full Text Available Milk fat is a key factor affecting milk quality and is also a major trait targeted in dairy cow breeding. To determine how the synthesis and the metabolism of lipids in bovine milk is regulated at the miRNA level, primary mammary epithelial cells (pMEC derived from two Chinese Holstein dairy cows that produced extreme differences in milk fat percentage were cultured by the method of tissue nubbles culture. Small RNA libraries were constructed from each of the two pMEC groups, and Solexa sequencing and bioinformatics analysis were then used to determine the abundance of miRNAs and their differential expression pattern between pMECs. Target genes and functional prediction of differentially expressed miRNAs by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis illustrated their roles in milk fat metabolism. Results show that a total of 292 known miRNAs and 116 novel miRNAs were detected in both pMECs. Identification of known and novel miRNA candidates demonstrated the feasibility and sensitivity of sequencing at the cellular level. Additionally, 97 miRNAs were significantly differentially expressed between the pMECs. Finally, three miRNAs including bta-miR-33a, bta-miR-152 and bta-miR-224 whose predicted target genes were annotated to the pathway of lipid metabolism were screened and verified by real-time qPCR and Western-blotting experiments. This study is the first comparative profiling of the miRNA transcriptome in pMECs that produce different milk fat content.

  5. Mechanisms of uptake of norepinephrine (NE) and Meta-iodobenzylguanidine (MIBG) into cultured bovine adrenomedullary cells: Evidence for uptake-one

    International Nuclear Information System (INIS)

    The nature of the sodium-dependent and sodium-independent uptake systems for NE and MIBG were studied in cultured bovine adrenomedullary cells, a model system exhibiting uptake mechanisms analogous to those in human pheochromocytomas. At concentrations ≤1.0μM, uptake of NE and MIBG was primarily by the sodium-dependent system; at higher concentrations, ≥ 20 μM, uptake of both agents was predominantly by the sodium-independent pathway. Desmethylimipramine (DMI) inhibited the sodium-dependent uptake of 0.5 and 20.0 μM NE (IC-50 = 0.05 and 1.0 μM, respectively) and the sodium-dependent uptake of 0.5 μM MIBG(IC-50 of 0.05 μM). However, at 0.5 and 20 μM, NE and MIBG, the sodium-independent uptake was 100 to 1000 fold less sensitive to the inhibitory effects of DMI. Cocaine, an Uptake-one inhibitor structurally unrelated to DMI, inhibited the sodium-dependent and independent NE and MIBG uptake in a pattern similar to DMI. DMI was a competitive inhibitor of sodium-dependent NE and MIBG uptake but not of sodium-independent uptake. Similarly, NE and MIBG were competitive inhibitors of each other when uptake occurred by the sodium-dependent pathway but not when uptake occurred by the sodium-independent pathway. The sodium-dependent pathway is a saturable, energy-dependent system which is inhibited by classical Uptake-one inhibitors. The sodium-independent uptake system may represent a diffusion mechanism. Consistent with these in vitro results, scintiscans of pheochromocytomas with [I-131]MIBG have been altered in vivo by DMI-like drugs and, therefore, appear to reflect Uptake-one

  6. Expression Profile of Genes as Indicators of Developmental Competence and Quality of In Vitro Fertilization and Somatic Cell Nuclear Transfer Bovine Embryos

    Science.gov (United States)

    Monteleone, Melisa Carolina; Mucci, Nicolas; Kaiser, German Gustavo; Brocco, Marcela; Mutto, Adrián

    2014-01-01

    Reproductive biotechnologies such as in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA) of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70), endoplasmic reticulum (ER) stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5) and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3) in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A) + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip) was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART). PMID:25269019

  7. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos.

    Directory of Open Access Journals (Sweden)

    Maria Jesús Cánepa

    Full Text Available Reproductive biotechnologies such as in vitro fertilization (IVF and somatic cell nuclear transfer (SCNT enable improved reproductive efficiency of animals. However, the birth rate of in vitro-derived embryos still lags behind that of their in vivo counterparts. Thus, it is critical to develop an accurate evaluation and prediction system of embryo competence, both for commercial purposes and for scientific research. Previous works have demonstrated that in vitro culture systems induce alterations in the relative abundance (RA of diverse transcripts and thus compromise embryo quality. The aim of this work was to analyze the RA of a set of genes involved in cellular stress (heat shock protein 70-kDa, HSP70, endoplasmic reticulum (ER stress (immunoglobulin heavy chain binding protein, Bip; proteasome subunit β5, PSMB5 and apoptosis (BCL-2 associated X protein, Bax; cysteine aspartate protease-3, Caspase-3 in bovine blastocysts produced by IVF or SCNT and compare it with that of their in vivo counterparts. Poly (A + mRNA was isolated from three pools of 10 blastocysts per treatment and analyzed by real-time RT-PCR. The RA of three of the stress indicators analyzed (Bax, PSMB5 and Bip was significantly increased in SCNT embryos as compared with that of in vivo-derived blastocysts. No significant differences were found in the RA of HSP70 and Caspase-3 gene transcripts. This study could potentially complement morphological analyses in the development of an effective and accurate technique for the diagnosis of embryo quality, ultimately aiding to improve the efficiency of assisted reproductive techniques (ART.

  8. The pro-inflammatory cytokine tumor necrosis factor α stimulates expression of the carnitine transporter OCTN2 (novel organic cation transporter 2) and carnitine uptake via nuclear factor-κB in Madin-Darby bovine kidney cells.

    Science.gov (United States)

    Zhou, X; Ringseis, R; Wen, G; Eder, K

    2015-06-01

    Carnitine uptake into tissues is mediated mainly by the novel organic cation transporter 2 (OCTN2), whose expression is upregulated in the liver of early-lactating dairy cows. It has been shown recently that pro-inflammatory cytokines, including tumor necrosis factor α (TNFα), stimulate OCTN2 expression and carnitine uptake in intestinal cells and inflamed intestinal mucosa. Given that many early-lactating dairy cows show typical signs of hepatic and systemic inflammation, such as elevated concentrations of circulating TNFα and activation of the key regulator of inflammation, nuclear factor κB (NF-κB), in tissues, it is possible that upregulation of OCTN2 and increase of carnitine uptake by TNFα is mediated by NF-κB, a mechanism that might contribute to the upregulation of OCNT2 in the liver of early-lactating dairy cows. Thus, in the present study, we tested the hypothesis that TNFα stimulates OCTN2 gene expression and carnitine uptake via NF-κB in the bovine Madin-Darby bovine kidney (MDBK) cell line. Treatment with TNFα caused activation of NF-κB, increased the mRNA and protein concentration of OCTN2, and stimulated the uptake of carnitine in MDBK cells. In contrast, combined treatment of MDBK cells with TNFα and the NF-κB inhibitor BAY 11-7085 completely blocked the effect of TNFα on OCTN2 mRNA and protein concentration and uptake of carnitine. These findings suggest that the bovine OCTN2 gene and carnitine uptake are regulated by NF-κB. Future studies are required to show the in vivo relevance of this regulatory mechanism in cattle.

  9. Deep sequencing-based transcriptional analysis of bovine mammary epithelial cells gene expression in response to in vitro infection with Staphylococcus aureus stains.

    Directory of Open Access Journals (Sweden)

    Xiao Wang

    Full Text Available Staphylococcus aureus (S. aureus is an important etiological organism in chronic and subclinical mastitis in lactating cows. Given the fundamental role the primary bovine mammary epithelial cells (pBMECs play as a major first line of defense against invading pathogens, their interactions with S. aureus was hypothesized to be crucial to the establishment of the latter's infection process. This hypothesis was tested by investigating the global transcriptional responses of pBMECs to three S. aureus strains (S56,S178 and S36 with different virulent factors, using a tag-based high-throughput transcriptome sequencing technique. Approximately 4.9 million total sequence tags were obtained from each of the three S. aureus-infected libraries and the control library. Referenced to the control, 1720, 219, and 427 differentially expressed unique genes were identified in the pBMECs infected with S56, S178 and S36 S. aureus strains respectively. Gene ontology (GO and pathway analysis of the S56-infected pBMECs referenced to those of the control revealed that the differentially expressed genes in S56-infected pBMECs were significantly involved in inflammatory response, cell signalling pathways and apoptosis. In the same vein, the clustered GO terms of the differentially expressed genes of the S178-infected pBMECs were found to comprise immune responses, metabolism transformation, and apoptosis, while those of the S36-infected pBMECs were primarily involved in cell cycle progression and immune responses. Furthermore, fundamental differences were observed in the levels of expression of immune-related genes in response to treatments with the three S. aureus strains. These differences were especially noted for the expression of important pro-inflammatory molecules, including IL-1α, TNF, EFNB1, IL-8, and EGR1. The transcriptional changes associated with cellular signaling and the inflammatory response in this study may reflect different immunomodulatory mechanisms

  10. Bovine colostrum enhances natural killer cell activity and immune response in a mouse model of influenza infection and mediates intestinal immunity through toll-like receptors 2 and 4.

    Science.gov (United States)

    Wong, Eric B; Mallet, Jean-François; Duarte, Jairo; Matar, Chantal; Ritz, Barry W

    2014-04-01

    Oral administration of bovine colostrum affects intestinal immunity, including an increased percentage of natural killer (NK) cells. However, effects on NK cell cytotoxic activity and resistance to infection as well as a potential mechanism remain unclear. Therefore, we investigated the effects of bovine colostrum (La Belle, Inc, Bellingham, WA) on the NK cytotoxic response to influenza infection and on toll-like receptor (TLR) activity in a primary intestinal epithelial cell culture. We hypothesized that colostrum would increase NK cell activity and that TLR-2 and TLR-4 blocking would reduce interleukin 6 production by epithelial cells in response to contact stimulation with colostrum. Four-month-old female C57BL/6 mice were supplemented with 1 g of colostrum per kilogram of body weight before and after infection with influenza A virus (H1N1). Animals were assessed for weight loss, splenic NK cell activity, and lung virus titers. Colostrum-supplemented mice demonstrated less reduction in body weight after influenza infection, indicating a less severe infection, increased NK cell cytotoxicity, and less virus burden in the lungs compared with controls. Colostrum supplementation enhanced NK cell cytotoxicity and improved the immune response to primary influenza virus infection in mice. To investigate a potential mechanism, a primary culture of small intestine epithelial cells was then stimulated with colostrum. Direct activation of epithelial cells resulted in increased interleukin 6 production, which was inhibited with TLR-2 and TLR-4 blocking antibodies. The interaction between colostrum and immunity may be dependent, in part, on the interaction of colostrum components with innate receptors at the intestinal epithelium, including TLR-2 and TLR-4. PMID:24774068

  11. Cartilage (Bovine and Shark) (PDQ)

    Science.gov (United States)

    ... Ask about Your Treatment Research Cartilage (Bovine and Shark) (PDQ®)–Patient Version Overview Go to Health Professional ... 8 ). Questions and Answers About Cartilage (Bovine and Shark) What is cartilage? Cartilage is a type of ...

  12. Lingual antimicrobial peptide and lactoferrin concentrations and lactoperoxidase activity in bovine colostrum are associated with subsequent somatic cell count.

    Science.gov (United States)

    Isobe, Naoki; Shibata, Ayumi; Kubota, Hirokazu; Yoshimura, Yukinori

    2013-11-01

    The present study was undertaken to examine whether potential levels of innate immune factors (lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO)) in colostrum are associated with subsequent milk somatic cell count (SCC) in dairy cows. Quarter milk samples were collected daily for 1 week postpartum to measure LAP and LF concentrations and LPO activity. SCC in milk was determined weekly for 2 months postpartum and its correlations to concentrations of LAP and LF and LPO activity were examined. Only small variations of all immune factors were found among four udders in each individual cow, whereas there were great differences in these factors among cows. Negative correlation was detected only between LPO activity and mean and maximum SCC, whereas its relationship was not significant. LAP and LF concentrations were significantly correlated positively to mean, maximum and minimum SCC. These results suggest that the great difference in innate immune factors among animals and high LAP and LF concentrations in colostrum may be associated with subsequent high incidence of SCC increase. PMID:24001397

  13. Lingual antimicrobial peptide and lactoferrin concentrations and lactoperoxidase activity in bovine colostrum are associated with subsequent somatic cell count.

    Science.gov (United States)

    Isobe, Naoki; Shibata, Ayumi; Kubota, Hirokazu; Yoshimura, Yukinori

    2013-11-01

    The present study was undertaken to examine whether potential levels of innate immune factors (lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO)) in colostrum are associated with subsequent milk somatic cell count (SCC) in dairy cows. Quarter milk samples were collected daily for 1 week postpartum to measure LAP and LF concentrations and LPO activity. SCC in milk was determined weekly for 2 months postpartum and its correlations to concentrations of LAP and LF and LPO activity were examined. Only small variations of all immune factors were found among four udders in each individual cow, whereas there were great differences in these factors among cows. Negative correlation was detected only between LPO activity and mean and maximum SCC, whereas its relationship was not significant. LAP and LF concentrations were significantly correlated positively to mean, maximum and minimum SCC. These results suggest that the great difference in innate immune factors among animals and high LAP and LF concentrations in colostrum may be associated with subsequent high incidence of SCC increase.

  14. Characterization of Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) interaction with the Bovine Aortic Endothelial (BAE) cell surface : Examination of the Role of Heparan Sulfate Proteoglycans (HSPG).

    OpenAIRE

    Parghi, Nirav

    1998-01-01

    Insulin-like growth factor binding proteins (IGFBPs) are known to be important modulators of the insulin-like growth factor (IGF-I). However, their precise role is as yet unclear. Further, recent studies have indicated that IGFBP-3 has a receptor mediated growth inhibitory response of its own. In the present study, we quantified the binding characteristics of IGFBP-3 to bovine aortic endothelial (BAE) cells. Binding studies at 4 oC were conducted and a specific binding curve for IGFB...

  15. Transactivation-Competent Bovine Papillomavirus E2 Protein Is Specifically Required for Efficient Repression of Human Papillomavirus Oncogene Expression and for Acute Growth Inhibition of Cervical Carcinoma Cell Lines

    OpenAIRE

    Goodwin, Edward C.; Naeger, Lisa Kay; Breiding, David E.; Androphy, Elliot J.; DiMaio, Daniel

    1998-01-01

    The papillomavirus E2 proteins can function as sequence-specific transactivators or transrepressors of transcription and as cofactors in viral DNA replication. We previously demonstrated that acute expression of the bovine papillomavirus type 1 (BPV1) E2 protein in HeLa and HT-3 cervical carcinoma cell lines greatly reduced cellular proliferation by imposing a specific G1/S phase growth arrest. In this report, we analyzed the effects of a panel of point mutations in the BPV1 E2 protein to ide...

  16. Correlation between circulating white blood cell counts and level of protective immune response against bovine viral diarrhea virus elicited by a modified live vaccine

    Science.gov (United States)

    Two trials (T1 and T2) were conducted to examine the range of responses elicited against bovine viral diarrhea virus (BVDV) by vaccination with modified live vaccine and to determine the level of response required for prevention of clinical disease. For T1, BVDV neutralizing (BVDV VN) titers were de...

  17. 77 FR 15847 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

    Science.gov (United States)

    2012-03-16

    ..., ``Analysis of Bovine Spongiform Encephalopathy (BSE) Risk to the U.S. Cattle Population from Importation of... final rule did not limit the importation of bovine-derived meat from Canada to that derived from cattle... meat from bovines 30 months of age or older while continuing to prohibit the importation of live...

  18. Ultrastructural characterization of bovine umbilical cord blood cells Caracterização ultra-estrutural das células sanguíneas do cordão umbilical bovino

    Directory of Open Access Journals (Sweden)

    Gustavo C Rodrigues

    2010-10-01

    Full Text Available The umbilical cord blood (UCB is an important source of pluripotent stem cells, which motivated researches on ontogeny and transplantation. The morphological characterization of umbilical cord cells is the first step to establish subsequent experiments on these areas. Although some information on humans can be found, no data on UCB is available for bovines. Therefore, this work is the first attempt to conduct an ultrastructural characterization of bovine umbilical cord blood. Blood was collected from the umbilical cord of twenty fetuses by punction of the umbilical vein. Samples were processed for whole leucocytes observation by centrifugation and the buffy coat was collected. Cells were washed and pelleted and prepared according to the standard protocol of the transmission electron microscopy. The presence of cells with morphologic characteristics compatible with the precursors from the erythrocytic, neutrophilic, eosinophilic, basophilic, and lymphocytic lineages was observed. Atypical cells with peculiar morphological features, strongly similar to apoptotic cells, were seen. Bovine neutrophils with three types of cytoplasmic granules were also found in the blood. The ultrastructural characteristics of observed bovine UCB cells where similar to those found in other species, suggesting that bovines could possibly constitute an experimental model for approaches on UCB cells research.O sangue de cordão umbilical (SCU é uma importante fonte de células progenitoras pluripotentes, que motiva pesquisas em ontogenia e transplantes. A caracterização morfológica das células de cordão umbilical é o primeiro passo para se estabelecer experimentos subsequentes nessas áreas. Embora algumas informações sobre SCU em humanos possam ser encontradas, não existe nenhuma informação disponível sobre elas em bovinos. Portanto, este trabalho é a primeira tentativa de se conduzir uma caracterização ultra-estrutural do sangue de cordão umbilical

  19. 利用piggyBac转座子制备牛成体多能干细胞诱导技术研究%Study on Generation of Bovine Induced Pluripotent Stem Cells Treated by piggyBac

    Institute of Scientific and Technical Information of China (English)

    赵丽霞; 张金吨; 张健; 李云霞; 苏杰; 孙伟; 赵高平; 戴雁峰; 郭继彤

    2013-01-01

    通过逆转录病毒等媒介表达核转录因子Oct4、Sox2、Klf4、c-Myc可将体细胞重编程为诱导多能干细胞(induced pluripotent stem cells,iPSc).时至今日,已经报道了小鼠、人、大鼠、猪、羊、马、牛的iPS细胞,但大动物iPS的多能性特别是嵌合体形成和生殖细胞传代还没有得到确认.与逆转录病毒等不同的是,piggyBac转座子转染效率高且无病毒源性、操作简单,可以在转座酶的存在下被安全切除.首次尝试了采用piggyBac转座子携带鼠源Oct4、Sox2、Klf4、c-Myc、Rarg和Lrh1 6个核转录因子诱导胎牛成纤维细胞,成功获得牛类iPS细胞,其形态与小鼠胚胎干细胞相似,克隆边界清晰、呈丘状、克隆内细胞致密、核大.RT-PCR与免疫组织化学染色分析均显示牛类iPS细胞表达多能性基因.该类细胞体外诱导分化可形成类胚体EB,且表达3个胚层的基因;体内诱导分化可形成畸胎瘤,苏木精、伊红染色显示瘤体有三胚层的分化.上述结果显示利用piggyBac转座子制备牛多潜能干细胞诱导技术可行,产生的牛类iPS细胞具有潜在多能性.%Authentic bovine embryonic stem cells have not been established despite over two decades efforts. Recently, induced pluripotent stem cells ( iPSCs) derived from somatic cells have been successfully generated from mouse, human, monkey, rat, pig, sheep, horse and cow. However, the pluripotency of large domestic animal iPS cells have not yet been tested by generation of chimera and germline transmission. Differently to virus vector, piggyback transposon was used to reprogram bovine fetal fibroblast (BFF) cells into iPS like cells. The piggyback transposon and transposase system is simple N virus-free and efficient. It just need transposase enzyme to catalyse insertion or excision events. It was reported that coexpress mouse Rarg (RAR-y) and Lrh-1 (liver receptor homologue 1) with the four factors Oct4、Sox2、Klf4 and c-Myc can obtain

  20. Bovine rhinitis viruses are common in U.S. cattle with bovine respiratory disease.

    Science.gov (United States)

    Hause, Ben M; Collin, Emily A; Anderson, Joe; Hesse, Richard A; Anderson, Gary

    2015-01-01

    Bovine rhinitis viruses (BRV) are established etiological agents of bovine respiratory disease complex however little research into their epidemiology and ecology has been published for several decades. In the U.S., only bovine rhinitis A virus 1 (BRAV1) has been identified while bovine rhinitis A virus 2 (BRAV2) and bovine rhinitis B virus (BRBV) were previously only identified in England and Japan, respectively. Metagenomic sequencing of a nasal swab from a bovine respiratory disease (BRD) diagnostic submission from Kansas identified contigs with approximately 90% nucleotide similarity to BRAV2 and BRBV. A combination of de novo and templated assemblies using reference genomes yielded near complete BRAV2 and BRBV genomes. The near complete genome of bovine rhinitis A virus 1 (BRAV1) was also determined from a historical isolate to enable further molecular epidemiological studies. A 5'-nuclease reverse transcription PCR assay targeting the 3D polymerase gene was designed and used to screen 204 archived BRD clinical specimens. Thirteen (6.4%) were positive. Metagenomic sequencing of six positive samples identified mixed BRAV1/BRAV2, BRAV1/BRBV and BRAV2/BRBV infections for five samples. One sample showed infection only with BRAV1. Seroprevalence studies using a cell culture adapted BRBV found immunofluorescence assay-reactive antibodies were common in the herds analyzed. Altogether, these results demonstrate that BRV infections are common in cattle with respiratory disease and that BRAV1, BRAV2 and BRBV co-circulate in U.S. cattle and have high similarity to viruses isolated more than 30 years ago from diverse locations.

  1. Bovine Spongiform Encephalopathy

    Science.gov (United States)

    Bovine spongiform encephalopathy (BSE), also referred to as “mad cow disease” is a chronic, non-febrile, neuro-degenerative disease affecting the central nervous system. The transmissible spongiform encephalopathies (TSEs) of domestic animals, of which BSE is a member includes scrapie of sheep...

  2. Bovine milk exosome proteome

    Science.gov (United States)

    Exosomes are 40-100 nm membrane vesicles of endocytic origin and are found in blood, urine, amniotic fluid, bronchoalveolar lavage (BAL) fluid, as well as human and bovine milk. Exosomes are extracellular organelles important in intracellular communication/signaling, immune function, and biomarkers ...

  3. Bovine Papillomavirus Clastogenic Effect Analyzed in Comet Assay

    OpenAIRE

    Araldi, R. P.; Melo, T. C.; N. Diniz; J. Mazzuchelli-de-Souza; R.F. Carvalho; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomavirus (BPV) is an oncogenic virus related to serious livestock diseases. Oncoproteins encoded by BPV are involved in several steps of cellular transformation and have been reported as presenting clastogenic effects in peripheral lymphocytes and primary culture cells. The aim of this study was to evaluate the clastogenic potential of BPV types 1, 2, and 4 by comet assay. Peripheral blood was collected from 37 bovines, 32 infected with different levels of papillomatosis (12 anim...

  4. Characterization of the bovine ampkgamma1 gene.

    Science.gov (United States)

    Benkel, Bernhard; Kollers, Sonja; Fries, Ruedi; Sazanov, Alexei; Yoshida, Erin; Valle, Edith; Davoren, Jon; Hickey, Donal

    2005-03-01

    AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals. AMPK plays a major role in protecting mammalian cells from metabolic stress by switching off biosynthetic pathways that require ATP and switching on ATP-regenerating pathways. In this report, we describe the isolation and characterization of the gene for the noncatalytic bovine gamma1 subunit of AMPK. The bovine ampkgamma1 (PRKAG1) gene spans in excess of 14 kb and is located at BTA 5q21-q22. It consists of 12 exons ranging in size from 38 b to 166 b, interspersed with 11 introns that range between 97 b and 6753 b in length. The coding region of the bovine gene shares 93% and 90% nucleotide sequence similarity with its human and rat counterparts, and the bovine AMPKgamma1 protein is 98% and 95% identical to its human and rat homologs, respectively, in amino acid sequence. SNP discovery using a cattle DNA panel revealed a number of polymorphisms that may be useful for the evaluation of ampkgamma1 as a candidate gene for energy metabolism-related production traits.

  5. Arginine Supplementation Recovered the IFN-γ-Mediated Decrease in Milk Protein and Fat Synthesis by Inhibiting the GCN2/eIF2α Pathway, Which Induces Autophagy in Primary Bovine Mammary Epithelial Cells.

    Science.gov (United States)

    Xia, Xiaojing; Che, Yanyi; Gao, Yuanyuan; Zhao, Shuang; Ao, Changjin; Yang, Hongjian; Liu, Juxiong; Liu, Guowen; Han, Wenyu; Wang, Yuping; Lei, Liancheng

    2016-05-31

    During the lactation cycle of the bovine mammary gland, autophagy is induced in bovine mammary epithelial cells (BMECs) as a cellular homeostasis and survival mechanism. Interferon gamma (IFN-γ) is an important antiproliferative and apoptogenic factor that has been shown to induce autophagy in multiple cell lines in vitro. However, it remains unclear whether IFN-γ can induce autophagy and whether autophagy affects milk synthesis in BMECs. To understand whether IFN-γ affects milk synthesis, we isolated and purified primary BMECs and investigated the effect of IFN-γ on milk synthesis in primary BMECs in vitro. The results showed that IFN-γ significantly inhibits milk synthesis and that autophagy was clearly induced in primary BMECs in vitro within 24 h. Interestingly, autophagy was observed following IFN-γ treatment, and the inhibition of autophagy can improve milk protein and milk fat synthesis. Conversely, upregulation of autophagy decreased milk synthesis. Furthermore, mechanistic analysis confirmed that IFN-γ mediated autophagy by depleting arginine and inhibiting the general control nonderepressible-2 kinase (GCN2)/eukaryotic initiation factor 2α (eIF2α) signaling pathway in BMECs. Then, it was found that arginine supplementation could attenuate IFN-γ-induced autophagy and recover milk synthesis to some extent. These findings may not only provide a novel measure for preventing the IFN-γ-induced decrease in milk quality but also a useful therapeutic approach for IFN-γ-associated breast diseases in other animals and humans.

  6. Evaluation of biofilm formation using milk in a flow cell model and microarray characterization of Staphylococcus aureus strains from bovine mastitis.

    Science.gov (United States)

    Snel, G G M; Malvisi, M; Pilla, R; Piccinini, R

    2014-12-01

    It was hypothesized that biofilm could play an important role in the establishment of chronic Staphylococcus aureus bovine mastitis. The in vitro evaluation of biofilm formation can be performed either in closed/static or in flow-based systems. Efforts have been made to characterize the biofilm-forming ability of S. aureus mastitis isolates, however most authors used static systems and matrices other than UHT milk. It is not clear whether such results could be extrapolated to the mammary gland environment. Therefore, the present study aimed to investigate the biofilm-forming ability of S. aureus strains from subclinical bovine mastitis using the static method and a flow-based one. One hundred and twelve strains were tested by the classic tissue culture plate assay (TCP) and 30 out of them were also tested by a dynamic semi-quantitative assay using commercial UHT milk as culture medium (Milk Flow Culture, MFC) or Tryptic Soy Broth as control medium (TS Flow Culture, TSFC). Only 6 (20%) strains formed biofilm in milk under flow conditions, while 36.6% were considered biofilm-producers in TCP, and 93.3% produced biofilm in TSFC. No agreement was found between TCP, MFC and TSFC results. The association between strain genetic profile, determined by microarray, and biofilm-forming ability in milk was evaluated. Biofilm formation in MFC was significantly associated with the presence of those genes commonly found in bovine-associated strains, assigned to clonal complexes typically detected in mastitis. Based on our results, biofilm-forming potential of bovine strains should be critically analysed and tested applying conditions similar to mammary environment. PMID:25448449

  7. Bovine Insulin Filaments Induced by Reducing Disulfide Bonds Show a Different Morphology, Secondary Structure, and Cell Toxicity from Intact Insulin Amyloid Fibrils

    OpenAIRE

    Zako, Tamotsu; Sakono, Masafumi; Hashimoto, Naomi; Ihara, Masaki; Maeda, Mizuo

    2009-01-01

    Amyloid fibrils are associated with more than 20 diseases, including Alzheimer's disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed du...

  8. Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum.

    Science.gov (United States)

    Tachikawa, E; Nogimori, K; Takahashi, S; Mizuma, K; Itoh, K; Kashimoto, T; Nagaoka, Y; Iida, A; Fujita, T

    1996-06-13

    Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes. PMID:8679651

  9. Cytotoxicity evaluation of luting resin cements on bovine dental pulp-derived cells (bDPCs) by real-time cell analysis.

    Science.gov (United States)

    Arslan Malkoç, Meral; Demir, Necla; Şengün, Abdulkadir; Bozkurt, Şerife Buket; Hakki, Sema Sezen

    2015-01-01

    To evaluate the cytotoxicity of resin cements on dental pulp-derived cells (bDPCs), Bifix QM (BQM), Choice 2(C2), RelyX U200(RU200), Maxcem Elite(ME), and Multilink Automix(MA) were tested. The materials were incubated in DMEM for 72 h. A real-time cell analyzer was used to evaluate cell survival. The statistical analyses used were one-way ANOVA and Tukey-Kramer tests. BQM, RU200, and ME demonstrated a significant decrease in the bDPCs' index at 24 and 72 h (p≤0.001). These materials were found to be the most toxic resin cements, as compared to the control and other tested materials (C2 and MA). However, C2 and MA showed a better survival rate, compared to BQM, RU200, and ME, and had lower cell index than the control group. The cytotoxic effects of resin cements on pulpa should be evaluated during the selection of proper cements. PMID:25736260

  10. Experimental fetal infection with bovine viral diarrhea virus. II. Morphological reactions and distribution of viral antigen.

    OpenAIRE

    Ohmann, H B

    1982-01-01

    The effect of an infection with bovine viral diarrhea virus on fetal bovine tissues as well as the tissue-localization of viral antigen are described. Four bovine fetuses, 120-165 days of gestation, were inoculated in utero with a second passage virus strain. Lymphoid tissues were studied by light and electron microscopy. The infection induced precocious development of the secondary lymphoid organs. Characteristic changes were seen in postcapillary venules, cells of the mononuclear phagocyte ...

  11. Establishment of bovine prion peptide-based monoclonal antibodies for identifying bovine prion

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    To obtain high titer monoclonal antibodies(McAbs) which can react with mammalian prion protein(PrP),Balb/C mice were immunized with bovine(Bo) PrP peptide(BoPrP 209-228 aa) coupled to keyhole limpet hemocyanin(KLH).The hybridoma cell lines secreting monoclonal antibodies against the peptide were established by cell fusion and cloning.The obtained McAbs were applied to detect recombinant human,bovine and hamster PrP,cellular prion protein(PrPc) in normal bovine brain and pathogenic scrapie prion protein(PrPSc) accumulated in the medulla oblongata of bovine spongiform encephalopathy(BSE)specimen with Western blot and immunohistochemical detection,respectively.The current procedure might offer a simple,feasible method to raise high titer antibodies for studying biological features of PrP in mammals,as well as detection of transmissible spongiform encephalopathy(TSE) and diagnosis of BSE,in particular.

  12. Isolation and transfection of skeletal muscle satellite cells from Qinchuan fetal bovine%秦川牛胎儿骨骼肌卫星细胞的分离培养

    Institute of Scientific and Technical Information of China (English)

    何玉龙; 吴月红; 权富生; 刘琴; 张涌

    2012-01-01

    【目的】探索胎牛骨骼肌卫星细胞分离、培养、鉴定及基因转染的方法。【方法】分别采用Ⅰ型胶原酶消化、Ⅰ型胶原酶与胰蛋白酶二步消化及链霉蛋白酶消化法对胎牛骨骼肌卫星细胞进行培养,比较了3种不同分离方法所得细胞数及其存活率的差异;利用差速贴壁法和Percoll密度梯度离心相结合的方法纯化骨骼肌卫星细胞,对纯化后的细胞进行myostatin基因RT-PCR以及结蛋白(Desmin)免疫细胞化学染色鉴定,最后通过电转染法对纯化的骨骼肌卫星细胞进行EGFP基因转染研究。【结果】3种消化培养方法中,以链霉蛋白酶消化法分离得到的胎牛骨骼肌卫星细胞数显著高于其它2种方法(P〈0.05),但细胞存活率较低(P〈0.05);而采用Ⅰ型胶原酶与胰蛋白酶二步消化法可以得到相对较高的细胞数及存活率。利用差速贴壁和Percoll密度梯度离心相结合的方法可以得到纯化的骨骼肌卫星细胞;电转染法适用于骨骼肌卫星细胞的基因转染。【结论】建立了胎牛骨骼肌卫星细胞分离、培养、纯化、鉴定及基因转染的方法,为通过转基因方法改良秦川牛产肉性能研究奠定了基础。%【Objective】 The study was done to establish a stable method for isolation,purification and gene transfection of Qinchuan fetal bovine skeletal muscle satellite cells.【Method】 Fetal cattle muscle satellite cells were isolated from Semimembranosus muscle by digesting with collagenase Ⅰ,collagenase Ⅰ combined with trypsin(two-step methods) or pronase.The differences in cells number and survival rate were compared.The cells were purified by Percoll density gradient centrifugation combined with different adherent methods.The cells were characterized by RT-PCR for myostatin gene and immunocytochemistry for Desmin.Then EGFP gene was introduced by electric transfection method.【Result】 The results showed that the

  13. Hedgehog signaling pathway in small bovine ovarian follicles

    Science.gov (United States)

    The hedgehog signaling pathway is involved in the regulation of cell proliferation, differentiation, and turnover in a variety of mammalian embryonic and adult tissues including bovine ovarian granulosa and theca cells. Binding of hedgehog to the patch receptor derepresses smoothened resulting in t...

  14. Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella

    International Nuclear Information System (INIS)

    Current European legislation for the registration and authorisation of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processes of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility

  15. Bovine coronavirus hemagglutinin protein.

    Science.gov (United States)

    King, B; Potts, B J; Brian, D A

    1985-02-01

    Treatment of purified bovine coronavirus (Mebus strain) with pronase destroyed the integrity of virion surface glycoproteins gp140, gp120, gp100, reduced the amount of gp26 and destroyed the hemagglutinating activity of the virus. Bromelain, on the other hand, destroyed the integrity of gp120, gp100 and gp26 but failed to remove gp140 and failed to destroy viral hemagglutinating activity. These experiments suggest that gp140 is the virion hemagglutinin. Immunoblotting studies using monospecific antiserum demonstrate that gp140 is a disulfide-linked dimeric structure reducible to monomers of 65 kDa.

  16. Camel and bovine chymosin

    DEFF Research Database (Denmark)

    Jensen, Jesper Langholm; Mølgaard, Anne; Poulsen, Jens-Christian Navarro;

    2013-01-01

    Bovine and camel chymosin are aspartic peptidases that are used industrially in cheese production. They cleave the Phe105-Met106 bond of the milk protein κ-casein, releasing its predominantly negatively charged C-terminus, which leads to the separation of the milk into curds and whey. Despite...... chymosin. Both enzymes possess local positively charged patches on their surface that can play a role in interactions with the overall negatively charged C-terminus of κ-casein. Camel chymosin contains two additional positive patches that favour interaction with the substrate. The improved electrostatic...

  17. Bovine Virus Diarrhea (BVD)

    OpenAIRE

    Hoar, Bruce R.

    2004-01-01

    Bovine virus diarrhea (BVD) is a complicated disease to discuss as it can result in a wide variety of disease problems from very mild to very severe. BVD can be one of the most devastating diseases cattle encounter and one of the hardest to get rid of when it attacks a herd. The viruses that cause BVD have been grouped into two genotypes, Type I and Type II. The disease syndrome caused by the two genotypes is basically the same, however disease caused by Type II infection is often more severe...

  18. Carcinoma de células escamosas en linfonódulo mandibular diagnosticado a la inspección en matadero como tuberculosis bovina Squamous cell carcinoma in a mandibular lymph node diagnosed as bovine tuberculosis during slaughterhouse inspection

    Directory of Open Access Journals (Sweden)

    C Lecocq

    2012-01-01

    Full Text Available El presente trabajo establece que el diagnóstico de tuberculosis (TBC bovina comprende múltiples técnicas que en conjunto llegan a un diagnóstico certero, no obstante, la inspección en matadero es quizás una de las más relevantes en la vigilancia de la TBC bovina, por cuanto ésta debe abocarse a lo que indica el manual de inspección del Servicio Agrícola y Ganadero (SAG. Aún así, es posible encontrar interpretaciones erróneas de los hallazgos en matadero, que confunden al inspector y que instan a aumentar la rigurosidad de la inspección.The diagnosis of bovine tuberculosis (TBC involves the combination of several techniques to obtain an accurate diagnosis. Among them, the inspection of the animal body at the slaughterhouse is, perhaps, one of the most important for the surveillance of bovine TBC. In Chile, the guidelines for this practice appear in the Official Inspection Manual of the Agricultural and Livestock Service (SAG. Nevertheless, there are wrong interpretations of findings at the slaughterhouse. This study describes a squamous cell carcinoma in the submandibular lymph node of a cow that was initially diagnosed as tuberculosis, based on the presence of caseous necrosis and mineralization detected during postmortem gross examination. Squamous cell carcinoma is a common tumor of white-faced cows such as Hereford, which tend to metastasize to regional lymph nodes like the mandibular lymph node.

  19. Estimation of mean exocytic vesicle capacitance in mouse adrenal chromaffin cells

    OpenAIRE

    Moser, Tobias; Neher, Erwin

    1997-01-01

    Whole-cell membrane capacitance measurements are frequently used to monitor neuronal and nonneuronal secretory activity. However, unless individual fusion events can be resolved, the type of the fusing vesicles cannot be identified in these experiments. Here we apply statistical analysis of trial-to-trial variations between depolarization-induced capacitance increases of mouse adrenal chromaffin cells and obtain estimates for the capacitance contribution of individual exocytic vesicles betwee...