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Sample records for cultured bovine adrenal

  1. Angiotensin II binding to cultured bovine adrenal chromaffin cells: identification of angiotensin II receptors

    SciTech Connect

    Boyd, V.L.; Printz, M.P.

    1986-03-05

    Physiological experiments have provided evidence that angiotensin II stimulates catecholamine secretion from the adrenal gland. Their laboratory and others have now shown by receptor autoradiography the presence of angiotensin II receptors (AIIR) in bovine and rat adrenal medulla. In order to extend these studies they have undertaken to define AIIR on cultured bovine adrenal chromaffin cells. Cells were isolated using the method of Levitt including cell enrichment with Percoll gradient centrifugation. Primary cultures of bovine adrenal medullary cells were maintained in DME/F12 medium containing 10% FCS. Cells were characterized by immunocytochemistry for Met- and Leu-enkephalin, PNMT, DBH and Chromagranin A. Cultured cells bind with high affinity and specificity (/sup 125/I)-ANG II yielding a K/sub D/ of 0.74 nM and B/sub max/ of 24,350 sites/cell. After Percoll treatment values of .77 nm and 34,500 sites/cell are obtained. K/sub D/ values are in close agreement with that obtained in adrenal slices by Healy. Competition studies identify a rank order of binding by this receptor similar to that of other tissues. They conclude that cultured chromaffin cells provide a suitable model system for the investigation and characterization of the ANG II receptor and for cellular studies of its functional significance.

  2. Tetrodotoxin-insensitive Na+ channel activator palytoxin inhibits tyrosine uptake into cultured bovine adrenal chromaffin cells

    SciTech Connect

    Morita, K.; Teraoka, K.; Azuma, M.; Oka, M.; Hamano, S. )

    1991-07-01

    The effects of the tetrodotoxin-insensitive Na+ channel activator palytoxin on both the secretion of endogenous catecholamines and the formation of 14C-catecholamines from (14C)tyrosine were examined using cultured bovine adrenal chromaffin cells. Palytoxin was shown to cause the stimulation of catecholamine secretion in a concentration-dependent manner. However, this toxin caused the reduction rather than the stimulation of 14C-catecholamine formation at the same concentrations. Palytoxin failed to cause any alteration in the activity of tyrosine hydroxylase prepared from bovine adrenal medulla. Furthermore, the uptake of (14C)tyrosine into the cells was shown to be inhibited by this toxin under the conditions in which the suppression of 14C-catecholamine formation was observed, and this inhibitory action on tyrosine uptake was closely correlated with that on catecholamine formation. The inhibitory action of palytoxin on tyrosine uptake into the cells was observed to be noncompetitive, and this effect was not altered by the removal of Na+ from the incubation mixture. These results suggest that palytoxin may be able to inhibit the uptake of (14C)tyrosine into the cells, resulting in the suppression of 14C-catecholamine formation, probably through its direct action on the plasma membranes of bovine adrenal chromaffin cells.

  3. Upregulation of norepinephrine transporter function by prolonged exposure to nicotine in cultured bovine adrenal medullary cells.

    PubMed

    Itoh, Hideaki; Toyohira, Yumiko; Ueno, Susumu; Saeki, Satoru; Zhang, Han; Furuno, Yumi; Takahashi, Kojiro; Tsutsui, Masato; Hachisuka, Kenji; Yanagihara, Nobuyuki

    2010-09-01

    Nicotine acts on nicotinic acetylcholine receptors in the adrenal medulla and brain, thereby stimulating the release of monoamines such as norepinephrine (NE). In the present study, we examined the effects of prolonged exposure to nicotine on NE transporter (NET) activity in cultured bovine adrenal medullary cells. Treatment of adrenal medullary cells with nicotine increased [(3)H]NE uptake in both a time- (1-5 days) and concentration-dependent (0.1-10 muM) manner. Kinetic analysis showed that nicotine induced an increase in the V (max) of [(3)H]NE uptake with little change in K (m). This increase in NET activity was blocked by cycloheximide, an inhibitor of ribosomal protein synthesis, but not by actinomycin D, a DNA-dependent RNA polymerase inhibitor. [(3)H]NE uptake induced by nicotine was strongly inhibited by hexamethonium and mecamylamine but not by alpha-bungarotoxin, and was abolished by elimination of Ca(2+) from the culture medium. KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, attenuated not only nicotine-induced [(3)H]NE uptake but also (45)Ca(2+) influx in the cells. The present findings suggest that long-term exposure to nicotine increases NET activity through a Ca(2+)-dependent post-transcriptional process in the adrenal medulla.

  4. Enhancement by GABA of the stimulation-evoked catecholamine release from cultured bovine adrenal chromaffin cells.

    PubMed

    Kitayama, S; Morita, K; Dohi, T; Tsujimoto, A

    1990-05-01

    The possible involvement of GABAergic mechanisms in the catecholamine (CA) release from adrenal medulla was investigated in a primary culture of bovine adrenal chromaffin cells. GABA elicited CA release and enhanced acetylcholine (ACh)-, excess K(+)- and veratridine-evoked CA release. Muscimol, a selective GABAA receptor agonist, mimicked the action of GABA on CA release. On the other hand, baclofen, a GABAB receptor agonist, failed to affect basal or evoked CA release. Furthermore, bicuculline and picrotoxin blocked the enhancement by GABA of veratridine-evoked CA release without affecting basal CA release and CA release evoked by veratridine. In Ca2(+)-free medium, GABA failed to affect basal and caffeine-evoked CA release. ACh-evoked CA release was slightly reduced by bicuculline, whereas excess K(+)-evoked CA release was not, suggesting the involvement of endogenous GABA in CA release evoked by ACh. These results suggest a facilitatory modulation by GABA of basal and evoked release of CA from bovine adrenal medulla through GABAA receptor-mediated mechanisms.

  5. Monolayer co-culture of rat heart cells and bovine adrenal chromaffin paraneurons.

    PubMed

    Trifaró, J M; Tang, R; Novas, M L

    1990-04-01

    This paper describes a method for the preparation of co-cultures of rat heart cells and bovine adrenal chromaffin paraneurons. The most suitable condition for heart cell isolation was when a combination of trypsin-DNAse I in Locke's solution was used for digestion. The best co-culture conditions were obtained when 10(6) heart cells were plated on 7- to 8-d-old adrenal chromaffin paraneuron cultures containing 0.5 x 10(6) cells per 35-mm diameter culture dishes. Measurements of DNA (heart cells and chromaffin paraneurons), monitoring of beating frequency (heart cells), and catecholamine (chromaffin paraneurons) levels and release indicated that both cell types remain viable and functional for several weeks. Heart cells started their characteristic contractile activity 24 h earlier when plated either on viable or lysed chromaffin paraneurons, an effect apparently due to faster surface adhesion of heart cells. The beating frequency of heart cells increased after treatment of co-cultures with either noradrenaline or nicotine, with the latter agent acting indirectly through the release of chromaffin paraneuron catecholamines. Propranolol produced a dose-related inhibition of the responses to either noradrenaline or nicotine, thus suggesting that the increase in myocyte's beating activity was mediated through beta-receptors. Anti-myosin and anti-dopamine-beta-hydroxylase immunostaining was used for cell type identification and for the demonstration of body-to-body and process-to-process contacts between adrenal chromaffin paraneurons and heart cells. This co-culture system will serve as a starting point of further studies directed to understand a) the influence of a cell type on the development and on the phenotypic characteristics of a second cell type and b) the interaction of cells derived from different organs and species.

  6. Identification and characterization of an angiotensin II receptor on cultured bovine adrenal chromaffin cells

    SciTech Connect

    Boyd, V.L.

    1987-01-01

    The presence of an angiotensin II receptor on cultured bovine adrenal chromaffin cells was demonstrated by radioligand binding. A single class of finding sites with a K/sub D/ of 0.7 nM was characterized. The use of radioligands also allows the localization of receptors by autoradiography. Autoradiography demonstrated that approximately 50% of the isolated cells bound angiotensin II. It was of interest to see if angiotensin II bound to a cell that possessed a certain phenotype. In order to evaluate this possibility a technique was developed that combined autoradiography and immunocytochemistry. Results indicated that angiotensin II binding sites were not localized preferentially to either norepinephrine or epinephrine cells. Binding of angiotensin II was associated with the release of intracellular catecholamine stores. Cells were pre-loaded with /sup 3/H-norepinephrine and secretion was monitored by following radioactivity released into the supernatant. Alternatively, release of endogenous catecholamines was determined by fluorometric assay.

  7. Dual effects of nobiletin, a citrus polymethoxy flavone, on catecholamine secretion in cultured bovine adrenal medullary cells.

    PubMed

    Zhang, Han; Toyohira, Yumiko; Ueno, Susumu; Shinohara, Yuko; Itoh, Hideaki; Furuno, Yumi; Yamakuni, Tohru; Tsutsui, Masato; Takahashi, Kojiro; Yanagihara, Nobuyuki

    2010-08-01

    Nobiletin, a compound of polymethoxy flavones found in citrus fruits, possesses a wide range of pharmacological activities. Here we report the effects of nobiletin on catecholamine secretion in cultured bovine adrenal medullary cells. Nobiletin (1.0-100 microM) concentration-dependently stimulated catecholamine secretion and (45)Ca(2+) influx. Its stimulatory effect of nobiletin on catecholamine secretion was abolished by deprivation of extracellular Ca(2+) and partially inhibited by specific inhibitors of voltage-dependent Ca(2+) channels and Na(+)/Ca(2+) exchangers. On the other hand, nobiletin suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner. It also inhibited catecholamine secretion, (22)Na(+) influx and/or (45)Ca(2+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels. In Xenopus oocytes expressing alpha3beta4 neuronal acetylcholine receptors, nobiletin directly inhibited the current evoked by acetylcholine in a concentration-dependent manner similar to that observed in catecholamine secretion. The present findings suggest that nobiletin, by itself, stimulates catecholamine secretion via activation of voltage-dependent Ca(2+) channels or Na(+)/Ca(2+) exchangers, whereas it inhibits catecholamine secretion induced by acetylcholine through the suppression of Na(+) influx and Ca(2+) influx in cultured bovine adrenal medullary cells.

  8. Stimulatory actions of bioflavenoids on tyrosine uptake into cultured bovine adrenal chromaffin cells

    SciTech Connect

    Morita, K.; Hamano, S.; Oka, M.; Teraoka, K. )

    1990-09-28

    The effects of flavenoids on L-({sup 14}C)tyrosine uptake into cultured adrenal chromaffin cells were examined. Flavone markedly stimulated tyrosine uptake into these cells in a manner dependent on its concentration. Apigenin also caused a moderate stimulatory action, but quercetin had no significant effect on the uptake. Flavone also stimulated the uptake of histidine, but did not affect the uptake of serine, lysine, or glutamic acid. These results are considered to propose the possibility that flavonoids may be able to stimulate the precursor uptake into the cells, resulting in an enhancement of the biogenic amine production.

  9. Characterization of insulin-like growth factor I and insulin receptors on cultured bovine adrenal fasciculata cells. Role of these peptides on adrenal cell function

    SciTech Connect

    Penhoat, A.; Chatelain, P.G.; Jaillard, C.; Saez, J.M.

    1988-06-01

    We have characterized insulin-like growth factor I (IGF-I) and insulin receptors in cultured bovine adrenal cells by binding and cross-linking affinity experiments. At equilibrium the dissociation constant and the number of binding sites per cell for IGF-I were 1.4 +/- (SE) 0.3 x 10(-9) M and 19,200 +/- 2,100, respectively. Under reduction conditions, disuccinimidyl suberate cross-linked (/sup 125/I)iodo-IGF-I to one receptor complex with an Mr of 125,000. Adrenal cells also contain specific insulin receptors with an apparent dissociation constant (Kd) of 10(-9) M. Under reduction conditions (/sup 125/I)iodo-insulin binds to one band with an approximate Mr of 125,000. IGF-I and insulin at micromolar concentrations, but not at nanomolar concentrations, slightly stimulated DNA synthesis, but markedly potentiated the mitogenic action of fibroblast growth factor. Adrenal cells cultured in a serum-free medium containing transferrin, ascorbic acid, and insulin (5 micrograms/ml) maintained fairly constant angiotensin-II (A-II) receptor concentration per cell and increased cAMP release on response to ACTH and their steroidogenic response to both ACTH and A-II. When the cells were cultured in the same medium without insulin, the number of A-II receptors significantly decreased to 65% and the increased responsiveness was blunted. Treatment of such cells for 3 days with increasing concentrations of IGF-I (1-100 ng/ml) produced a 2- to 3-fold increase in A-II receptors and enhanced the cAMP response (3- to 4-fold) to ACTH and the steroidogenic response (4- to 6-fold) to ACTH and A-II. These effects were time and dose dependent (ED50 approximately equal to 10(-9) M). Insulin at micromolar concentrations produced an effect similar to that of IGF-I, but at nanomolar concentrations the effect was far less.

  10. Spontaneous and electrically-evoked catecholamine secretion from long-term cultures of bovine adrenal chromaffin cells.

    PubMed

    Noga, Brian R; Pinzon, Alberto

    2013-09-05

    Catecholamine release was measured from bovine adrenal medullary chromaffin cell (CC) cultures maintained over a period of three months. Cells were plated over simple biocompatible cell platforms with electrical stimulation capability and at specified times transferred to an acrylic superfusion chamber designed to allow controlled flow of superfusate over the culture. Catecholamine release was measured from the superfusates using fast cyclic voltammetry before, during and after electrical stimulation of the cells. Immunocytochemical staining of CC cultures revealed that they were composed of epinephrine (EP) and/or norepinephrine (NE) type cells. Both spontaneous and evoked-release of catecholamines from CCs were observed throughout the testing period. EP predominated during spontaneous release, whereas NE was more prevalent during electrically-evoked release. Electrical stimulation for 20 s, increased total catecholamine release by 60-130% (measured over a period of 500 s) compared to that observed for an equivalent 20 s period of spontaneous release. Stimulus intensity was correlated with the amount of evoked release, up to a plateau which was observed near the highest intensities. Shorter intervals between stimulation trials did not significantly affect the initial amount of release, and the amount of evoked release was relatively stable over time and did not decrease significantly with age of the culture. The present study demonstrates long-term survival of CC cultures in vitro and describes a technique useful for rapid assessment of cell functionality and release properties of cultured monoaminergic cell types that later can be transplanted for neurotransmitter replacement following injury or disease. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Halothane inhibits the cholinergic-receptor-mediated influx of calcium in primary culture of bovine adrenal medulla cells

    SciTech Connect

    Yashima, N.; Wada, A.; Izumi, F.

    1986-04-01

    Adrenal medulla cells are cholinoceptive cells. Stimulation of the acetylcholine receptor causes the influx of Ca to the cells, and Ca acts as the coupler of the stimulus-secretion coupling. In this study, the authors investigated the effects of halothane on the receptor-mediated influx of /sup 45/Ca using cultured bovine adrenal medulla cells. Halothane at clinical concentrations (0.5-2%) inhibited the influx of /sup 45/Ca caused by carbachol, with simultaneous inhibition of catecholamine secretion. The influx of /sup 45/Ca and the secretion of catecholamines caused by K depolarization were inhibited by a large concentration of Mg, which competes with Ca at Ca channels, but not inhibited by halothane. Inhibition of the /sup 45/Ca influx by halothane was not overcome by increase in the carbachol concentration. Inhibition of the /sup 45/Ca influx by halothane was examined in comparison with that caused by a large concentration of Mg by the application of Scatchard analysis as the function of the external Ca concentration. Halothane decreased the maximal influx of /sup 45/Ca without altering the apparent kinetic constant of Ca to Ca channels. On the contrary, a large concentration of Mg increased the apparent kinetic constant without altering the maximal influx of /sup 45/Ca. Based on these findings, the authors suggest that inhibition of the /sup 45/Ca influx by halothane was not due to the direct competitive inhibition of Ca channels, nor to the competitive antagonism of agonist-receptor interaction. As a possibility, halothane seems to inhibit the receptor-mediated activation of Ca channels through the interference of coupling between the receptor and Ca channels.

  12. Catecholamine release from cultured bovine adrenal medullary chromaffin cells in the presence of 60-Hz magnetic fields.

    PubMed

    Craviso, Gale L; Chatterjee, Indira; Publicover, Nelson G

    2003-04-01

    Effects of powerline frequency (50/60 Hz) electric and magnetic fields on the central nervous system may involve altered neurotransmitter release. This possibility was addressed by determining whether 60-Hz linearly polarized sinusoidal magnetic fields (MFs) alter the release of catecholamines from cultured bovine adrenal chromaffin cells, a well-characterized model of neural-type cells. Dishes of cells were placed in the center of each of two four-coil Merritt exposure systems that were enclosed within mu-metal chambers in matched incubators for simultaneous sham and MF exposure. Following 15-min MF exposure of the cells to flux densities of 0.01, 0.1, 1.0 or 2 mT, norepinephrine and epinephrine release were quantified by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. No significant differences in the release of either norepinephrine or epinephrine were detected between sham-exposed cells and cells exposed to MFs in either the absence or presence of Bay K-8644 (2 microM) or dimethylphenylpiperazinium (DMPP, 10 microM). Consistent with these null findings is the lack of effect of MF exposure on calcium influx. We conclude that catecholamine release from chromaffin cells is not sensitive to 60-Hz MFs at magnetic flux densities in the 0.01-2 mT range.

  13. Endothelial Cells from Bovine Adrenal Medulla Develop Capillary-Like Growth Patterns in Culture

    NASA Astrophysics Data System (ADS)

    Banerjee, Dipak K.; Ornberg, Richard L.; Youdim, Moussa B. H.; Heldman, Eli; Pollard, Harvey B.

    1985-07-01

    The endocrine barrier between chromaffin cells and the blood stream in the adrenal medulla is made of capillary endothelial cells. We have now succeeded in isolating endothelial cells from adrenal medullary tissue, which are probably derived from this barrier. These cells grow on plastic surfaces in the absence of special growth factors or collagen overlays and differentiate into organized structures quite similar to true capillaries. The cells contain factor VIII:R, a marker for endothelial cells, and form intercellular junctions characteristic of capillary endothelial cells. They also synthesize and secrete basal lamina structures and engage in transcytosis, a characteristic ultrastructural and functional combination of exocytosis and endocytosis across the thin endothelial cell processes. These endothelial cells can take up and deaminate catecholamines by A-type monoamine oxidase, an enzyme functionally distinct from the B-type monoamine oxidase found in chromaffin cells. These data indicate that the chromaffin cell and its endothelial cell neighbor may constitute the functional unit of catecholamine metabolism in the adrenal medulla.

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

    PubMed Central

    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 high K(+)-evoked increases in [Ca2+]i in a concentration-dependent manner without effect on the resting [Ca2+]i. 4. NKY-722 had little effect on basal release of noradrenaline and adrenaline but inhibited high K(+)-evoked release of noradrenaline and adrenaline in a concentration-dependent manner with IC50 values of 5.0 nM and 4.8 nM, respectively. 5. Nicardipine, a prototype of NKY-722, also inhibited high K(+)-evoked 45Ca2+ influx and release of noradrenaline and adrenaline in a concentration-dependent manner: the IC50 value for high K(+)-evoked 45Ca2+ influx was 51 nM, and the values for high K(+)-evoked release of noradrenaline and adrenaline were 52 nM and 50 nM, respectively. 6. These results show that NKY-722 is a hydrophilic Ca2+ antagonist ten times more potent than nicardipine. PMID:1912977

  15. Stimulatory effect of nobiletin, a citrus polymethoxy flavone, on catecholamine synthesis through Ser19 and Ser40 phosphorylation of tyrosine hydroxylase in cultured bovine adrenal medullary cells.

    PubMed

    Zhang, Han; Yanagihara, Nobuyuki; Toyohira, Yumiko; Takahashi, Keita; Inagaki, Hirohide; Satoh, Noriaki; Li, Xiaoja; Goa, Xiumei; Tsutsui, Masato; Takahaishi, Kojiro

    2014-01-01

    We previously reported the dual effects of nobiletin, a compound of polymethoxy flavones found in citrus fruits, on catecholamine secretion in cultured bovine adrenal medullary cells. Here, we report the effects of nobiletin on catecholamine synthesis in the cells. Nobiletin increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine in a time (20-30 min)- and concentration (1.0-100 μM)-dependent manner. Nobiletin (10-100 μM) also activated tyrosine hydroxylase activity. The stimulatory effect of nobiletin on (14)C-catecholamine synthesis was not observed when extracellular Ca(2+) was not present in the incubation medium. Protein kinase inhibitors including H-89, an inhibitor of cyclic AMP-dependent protein kinase, and KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, suppressed the stimulatory effects of nobiletin on catecholamine synthesis as well as tyrosine hydroxylase activity. Nobiletin also induced the phosphorylation of tyrosine hydroxylase at Ser(19) and Ser(40). Nobiletin (1.0-100 μM) inhibited (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that nobiletin, by itself, stimulates catecholamine synthesis through tyrosine hydroxylase phosphorylation at Ser(19) and Ser(40), whereas it inhibits catecholamine synthesis induced by acetylcholine in bovine adrenal medulla.

  16. Porcine brain natriuretic peptide receptor in bovine adrenal cortex

    SciTech Connect

    Higuchi, K.; Hashiguchi, T.; Ohashi, M.; Takayanagi, R.; Haji, M.; Matsuo, H.; Nawata, H.

    1989-01-01

    The action of porcine brain natriuretic peptide (pBNP) on the steroidogenesis was investigated in cultured bovine adrenocortical cells. Porcine BNP induced a significant dose-dependent inhibition of both ACTH- and A II-stimulated aldosterone secretion. 10/sup /minus/8/M and 10/sup /minus/7/M pBNP also significantly inhibited ACTH-stimulated cortisol and dehydroepiandrosterone (DHEA) secretions. Binding studies of (/sup 125/I)-pBNP to bovine adrenocortical membrane fractions showed that adrenal cortex had high-affinity and low-capacity pBNP binding sites, with a dissociation constant (Kd) of 1.70 x 10/sup /minus/10/M and a maximal binding capacity (Bmax) of 19.9 fmol/mg protein. Finally, the 135 Kd radioactive band was specially visualized in the affinity labeling of bovine adrenal cortex with disuccinimidyl suberate (DSS). These results suggest that pBNP may have receptor-mediated suppressive actions on bovine adrenal steroidogenesis, similar to that in atrial natriuretic peptide (ANP).

  17. Differential effects of short and prolonged exposure to carvedilol on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells.

    PubMed

    Kajiwara, Koji; Yanagita, Toshihiko; Nakashima, Yasuhide; Wada, Akihiko; Izumi, Futoshi; Yanagihara, Nobuyuki

    2002-07-01

    We examined the effects of short and prolonged exposure to carvedilol, an antihypertensive and beta-adrenoceptor blocking drug, on voltage-dependent Na(+) channels in cultured bovine adrenal medullary cells. Carvedilol (1-100 microM) reduced (22)Na(+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels. Carvedilol also suppressed veratridine-induced (45)Ca(2+) influx and catecholamine secretion in a concentration-dependent manner similar to that of (22)Na(+) influx. Prolonged exposure of the cells to 10 microM carvedilol increased [(3)H]saxitoxin ([(3)H]STX) binding, which reached a plateau at 12 h and was still observed at 48 to 72 h. Scatchard analysis of [(3)H]STX binding revealed that carvedilol increased the B(max) value (control, 14.9 +/- 0.9 fmol/10(6) cells; carvedilol, 23.8 +/- 1.2 fmol/10(6) cells) (n = 3, P < 0.05) without altering the K(d) value, suggesting a rise in the number of cell surface Na(+) channels. The increase in [(3)H]STX binding by carvedilol was prevented by cycloheximide, an inhibitor of protein synthesis, whereas carvedilol changed neither alpha- nor beta(1)-subunit mRNA levels of Na(+) channels. The carvedilol-induced increase of [(3)H]STX binding was abolished by brefeldin A and H-89, inhibitors of intracellular vesicular trafficking of proteins from the trans-Golgi network and of cyclic AMP-dependent protein kinase (protein kinase A), respectively. The present findings suggest that short-term treatment with carvedilol reduces the activity of Na(+) channels, whereas prolonged exposure to carvedilol up-regulates cell surface Na(+) channels. This may add new pharmacological effects of carvedilol to our understanding in the treatment of heart failure and hypertension.

  18. Histologic and immunohistochemical classification of 41 bovine adrenal gland neoplasms.

    PubMed

    Grossi, A B; Leifsson, P S; Jensen, H E; Vainer, B; Iburg, T

    2013-05-01

    Tumors of the adrenal glands are among the most frequent tumors in cattle; however, few studies have been conducted to describe their characteristics. The aim of this study was to classify 41 bovine adrenal neoplasms from 40 animals based on macroscopic and histologic examination, including electron microscopy and immunohistochemistry for melan A, synaptophysin, chromogranin A, vimentin, pan-cytokeratin, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase), and Ki-67. The tumors were classified as 23 adrenocortical adenomas, 12 adrenocortical carcinomas, 2 schwannomas, 2 pheochromocytomas (1 malignant), and 1 ganglioneuroma. Five histologic features were characteristic of metastasizing adrenocortical tumors: invasion of the capsule, vascular invasion, diffuse growth pattern, spindle-cell morphology, and nuclear pleomorphism. Adrenocortical tumors with at least 3 of these features were classified as malignant. Immunohistochemically, adrenocortical tumors expressed melan A (16/19), vimentin (14/26), cytokeratin (11/26), and chromogranin A (9/27), whereas pheochromocytomas expressed chromogranin A (2/2), synaptophysin (2/2), and vimentin (1/2). Both schwannomas expressed CNPase. An immunohistochemistry panel consisting of antibodies against melan A, synaptophysin, and CNPase was considered most useful to classify bovine adrenal tumors. However, the distinction between benign and malignant adrenocortical tumors was based on histologic features as in human medicine.

  19. Identification of D-2 dopaminergic receptors in bovine adrenal cortex

    SciTech Connect

    Missale, C.; Liberini, P.; Memo, M.; Carruba, M.O.; Spano, P.

    1985-12-30

    Dopamine receptors in bovine adrenal cortex have been studied by using /sup 3/H-(-) atsulpiride as selective ligand. The specific binding is saturable and the Scatchard analysis reveals a single component with a Kd of 6.2 nM and a Bmax of 8 fmoles/mg protein. The characterization indicates that the binding is rapid, reversible, stereospecific, Na/sup +/ - and temperature-dependent. Moreover its pharmacological profile is superimposable to that of D-2 receptors in the striatum, thus suggesting that central and peripheral D-2 receptors are identical. 27 references, 6 figures, 1 table.

  20. Alpha-human atrial natriuretic polypeptide (. cap alpha. -hANP) specific binding sites in bovine adrenal gland

    SciTech Connect

    Higuchi, K.; Nawata, H.; Kato, K.I.; Ibayashi, H.; Matsuo, H.

    1986-06-13

    The effects of synthetic ..cap alpha..-human atrial natriuretic polypeptide (..cap alpha..-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. ..cap alpha..-hANP did not inhibit basal aldosterone secretion. ..cap alpha..-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of aCTH (10/sup -8/M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of (/sup 125/I) ..cap alpha..-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of ..cap alpha..-hANP.

  1. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules

    SciTech Connect

    Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. ); Zahraoui, A.; Tavitian, A. )

    1990-08-01

    The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine {beta}-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

  2. Purification and characterization of bovine adrenal cytochrome b561 expressed in insect and yeast cell systems.

    PubMed

    Liu, Wen; Kamensky, Yury; Kakkar, Reva; Foley, Erin; Kulmacz, Richard J; Palmer, Graham

    2005-04-01

    Bovine adrenal chromaffin granule cytochrome (cyt) b561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-beta-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b561 in insect and yeast cell systems. The bovine cyt b561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (approximately 0.5 mg detergent-solubilized cyt b561/L culture). For the yeast system, the cyt b561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b561 expression (approximately 0.7 mg detergent-solubilized cyt b561/L culture). Recombinant His-tagged cyt b561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni-NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue.

  3. Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

    PubMed

    Santodomingo, Jaime; Vay, Laura; Camacho, Marcial; Hernández-Sanmiguel, Esther; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2008-10-01

    The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.

  4. Identification of pro-opiomelanocortin and secretion of its peptide fragments in bovine adrenals

    SciTech Connect

    Tennov, A.V.; Dmitriev, A.D.; Kizim, E.A.; Ustinova, E.E.

    1986-01-01

    This paper describes the results of an investigation to show that biosynthesis of POMC, its proteolytic processing, an secretion of the peptide products of that processing take place in the bovine adrenals. Rabbit antisera against endorphins were obtained and used for radioimmunoassay of peptides. I 125-labeled peptides were obtained by the chloramine method and purified from free I 125 on Sephadex G-10 (0.7 x 5 cm, centrifugation for 10 min at 1500 g). To detect secretion of peptide fragments of POMC in the adrenals experiments were undertaken to determine the beta-endorphin content in perfusates obtained during retrograde perfusion of the bovine adrenals. It was found that immunoreactive compounds, indistinguishable in their immunochemical properties from beta-endorphin, are present in the perfusates, just as in the tissue extracts.

  5. Inhibitory effect of strychnine on acetylcholine receptor activation in bovine adrenal medullary chromaffin cells.

    PubMed Central

    Kuijpers, G A; Vergara, L A; Calvo, S; Yadid, G

    1994-01-01

    1. Strychnine, which is known as a potent and selective antagonist of the inhibitory glycine receptor in the central nervous system, inhibits the nicotinic stimulation of catecholamine release from bovine cultured adrenal chromaffin cells in a concentration-dependent (1-100 microM) manner. At 10 microM nicotine, the IC50 value for strychnine is approximately 30 microM. Strychnine also inhibits the nicotine-induced membrane depolarization and increase in intracellular Ca2+ concentration. 2. The inhibitory action of strychnine is reversible and is selective for nicotinic stimulation, with no effect observed on secretion elicited by a high external K+ concentration, histamine or angiotensin II. 3. Strychnine competes with nicotine in its effect, but not modify the apparent positive cooperatively of the nicotine binding sites. In the absence of nicotine, strychnine has no effect on catecholamine release. Glycine does not affect catecholamine release nor the inhibitory action of strychnine on this release. 4. These results suggest that strychnine interacts with the agonist binding site of the nicotinic acetylcholine receptor in chromaffin cells, thus exerting a pharmacological effect independently of the glycine receptor. PMID:7834198

  6. Calcium gradients and exocytosis in bovine adrenal chromaffin cells.

    PubMed

    Marengo, Fernando D

    2005-08-01

    The relationship between the localized Ca(2+) concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca(2+) imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca(2+) gradients and small "synchronous" increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca(2+) concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and K(d) = 1.4 microM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca(2+) gradients collapsed. Raising the pipette Ca(2+) concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca(2+)-dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1-2 microM and vesicles are mobilized to this pool in a Ca(2+)-dependent manner.

  7. Mobile and immobile calcium buffers in bovine adrenal chromaffin cells.

    PubMed Central

    Zhou, Z; Neher, E

    1993-01-01

    1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble Ca2+ binding proteins in the range of 7-20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa S did not change in the range of Ca2+ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane

  8. Theoretical conformational analysis of the bovine adrenal medulla 12 residue peptide molecule

    NASA Astrophysics Data System (ADS)

    Akhmedov, N. A.; Tagiyev, Z. H.; Hasanov, E. M.; Akverdieva, G. A.

    2003-02-01

    The spatial structure and conformational properties of the bovine adrenal medulla 12 residue peptide Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Arg7-Val8-Gly9-Arg10-Pro11-Glu12 (BAM-12P) molecule were studied by theoretical conformational analysis. It is revealed that this molecule can exist in several stable states. The energy and geometrical parameters for the low-energy conformations are obtained. The conformationally rigid and labile segments of this molecule were revealed.

  9. Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

    SciTech Connect

    Fibbi, G.; Ziche, M.; Morbidelli, L. ); Magnelli, L.; Del Rosso, M. )

    1988-12-01

    On the basis of {sup 125}I-labeled plasminogen activator binding analysis the authors have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a K{sub d} of 0.8958{times}10{sup {minus}12} M. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.

  10. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    SciTech Connect

    Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.

    1988-05-15

    Prostaglandin E/sub 2/ (PEG/sub 2/) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its ..cap alpha.. subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G/sub 0/) purified from bovine brain. The molecular weight of the ..cap alpha.. subunit was 40,000, which is between those of G/sub i/ and G/sub 0/. The purified protein was also distinguished immunologically from G/sub i/ and G/sub 0/ and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles resulted in a remarkable restoration of (/sup 3/H)PGE/sub 2/ binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.

  11. Photoaffinity crosslinking of etorphine with opioid binding sites in the bovine adrenal medulla

    SciTech Connect

    Cantau, P.; Bourhim, N.; Giraud, P.; Oliver, C.; Castanas, E.

    1987-04-01

    The covalent crosslinking of (/sup 3/H)etorphine with opioid binding sites in the bovine adrenal medulla is reported. Of all the radiolabeled opiates tested (ethylketocyclazocine, etorphine, (D-Ala2, D-Leu5)enkephalin, (D-Ala2, Me-Phe4, Gly5-ol)enkephalin only etorphine could be crosslinked under uv irradiation. In our conditions (black uv lamp, 160 W, peak mean 360 nm, from a distance of 10 cm) maximum covalent binding was observed after a 10-min irradiation. Protein concentration was a crucial factor for the irreversible/total binding ratio. A good ratio (50%) was obtained at protein concentrations of about 1.0 mg/ml. Covalent binding of nonmodified opiates could be of interest for the biochemical characterization of their binding sites.

  12. Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules.

    PubMed Central

    Fleminger, G; Ezra, E; Kilpatrick, D L; Udenfriend, S

    1983-01-01

    Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla. PMID:6578517

  13. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  14. Metabolism of adrenic acid to vasodilatory 1alpha,1beta-dihomo-epoxyeicosatrienoic acids by bovine coronary arteries.

    PubMed

    Yi, Xiu-Yu; Gauthier, Kathryn M; Cui, Lijie; Nithipatikom, Kasem; Falck, John R; Campbell, William B

    2007-05-01

    Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by a two-carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06 +/- 0.01 and 6.18 +/- 0.60 microg/mg protein and adrenic acid concentrations of 0.29 +/- 0.01 and 1.56 +/- 0.16 microg/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid (10(-9)-10(-5) M) induced concentration-related relaxations (maximal relaxation = 83 +/- 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the K(+) channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 microM, maximal relaxation = 53 +/- 4%), and the cytochrome P-450 inhibitor, miconazole (10 microM, maximal relaxation = 52 +/- 5%). Reverse-phase HPLC and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxyeicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET [16,17-, 13,14-, 10,11-, and 7,8- (10(-9)-10(-5) M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 +/- 3%). Adrenic acid (10(-6) M) and DH-16,17-EET (10(-6) M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET (10(-8)-10(-6) M) activated iberiotoxin-sensitive, whole cell K(+) currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome P-450 metabolites. The adrenic acid metabolite, DH-16,17-EET, activates smooth muscle K(+) channels to cause hyperpolarization and

  15. Formation of inositol 1,3,4,6-tetrakisphosphate during angiotensin II action in bovine adrenal glomerulosa cells

    SciTech Connect

    Balla, T.; Guillemette, G.; Baukal, A.J.; Catt, K.J.

    1987-10-14

    Angiotensin II stimulates the formation of several inositol polyphosphates in cultured bovine adrenal glomerulosa cells prelabelled with (/sup 3/H) inositol. Analysis by high performance anion exchange chromatography of the inositol-phosphate compounds revealed the existence of two additional inositol tetrakisphosphate (InsP4) isomers in proximity to Ins-1,3,4,5-P4, the known phosphorylation product of Ins-1,4,5-trisphosphate and precursor of Ins-1,3,4-trisphosphate. Both of these new compounds showed a slow increase after stimulation with angiotensin II. The structure of one of these new InsP4 isomers, which is a phosphorylation product of Ins-1,3,4-P3, was deduced by its resistance to periodate oxidation to be Ins-1,3,4,6-P4. The existence of multiple cycles of phosphorylation-dephosphorylation reactions for the processing of Ins-1,4,5-P4 may represent a new aspect of the inositol-lipid related signalling mechanism in agonist-activated target cells.

  16. Bradykinin and histamine-induced cytosolic calcium increase in capillary endothelial cells of bovine adrenal medulla.

    PubMed

    Vinet, Raúl; Cortés, Magdalena P; Alvarez, Rocío; Delpiano, Marco A

    2014-09-01

    We have assessed the effect of bradykinin and histamine on the cytosolic free calcium concentration ([Ca(2+)]i ) of bovine adrenal medulla capillary endothelial cells (BAMCECs). To measure [Ca(2+)]i changes in BAMCECs the intracellular fluorescent probe, fluo-3 AM, was used. Bradykinin (3 µM) produced a transient monophasic increase in [Ca(2+)]i , which was depressed by B1650 (0.1 µM), a B2-bradykinin receptor antagonist (D-Arg-[Hyp(3), Thi(5,8) , D-Phe(7)]-Bradykinin). Similarly, increase in [Ca(2+)]i induced by histamine was also depressed by tripolidine (0.1 µM), an H1-histamine receptor antagonist. [Ca(2+)]i increase induced by both agonists was unaffected in the absence of extracellular Ca(2+) or presence of antagonists of voltage operated Ca(2+) channels (VOCCs). Thapsigargin (1 µM) did not abolish the increase of [Ca(2+)]i produced by bradykinin, but abolished that of histamine. In contrast, caffeine (100 µM), abolished the [Ca(2+)]i response induced by bradykinin (3 µM), but did not affect the [Ca(2+)]i increase induced by histamine (100 µM). The results indicate the presence of B2 bradykinin- and H1 histamine-receptors in BAMCECs. Liberation of Ca(2+) induced by both agonists occurs through 2 different intracellular mechanisms. While bradykinin activates a sarco(endo) plasmic reticulum (SER) containing a SER Ca(2+) -ATPase (SERCA) thapsigargin-insensitive, histamine activates a SER containing a SERCA thapsigargin-sensitive. We suggest that the increase in [Ca(2+)]i induced by bradykinin and histamine could be of physiological relevance, modulating adrenal gland microcirculation.

  17. Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

    PubMed Central

    Rondeau, M; Guay, P; Goff, A K; Cooke, G M

    1996-01-01

    The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows. PMID:8825988

  18. Characterization of angiotensin-binding sites in the bovine adrenal and the rat brain

    SciTech Connect

    Rogulja, I.

    1989-01-01

    The first study was designed to determine whether systemically administered MSG affects neurons in the CVOs that are potentially important in mediating angiotensin-dependent responses. Rats were pretreated with MSG and the receptors for angiotensin II were assayed by radioligand binding in brain homogenates from the septum anteroventral third ventricular region (AV3V) and the thalamus/hypothalamus region using {sup 125}I-angiotensin II as the radioligand. The results of this experiment indicate that systematically administered MSG in the rat significantly reduced the number (Bmax) of Ang II receptors in a tissue sample which contained both extra blood-brain barrier organs as well as tissue within the blood-brain barrier with no change in the affinity (Kd) of the binding sites. The second chapter reports the successful solubilization of bovine adrenal {sup 125}I Ang II and {sup 125}I Sar{sup 1},Ile{sup 8}-Ang II binding sites with the detergent CHAPS. The results of our studies indicate the presence of two angiotensin binding sites. The one site is specific for naturally occurring angiotensins as well as sarcosine-1 substituted angiotensin analogues. The other site which can be optimally stabilized be re-addition of 0.3% CHAPS into the incubation assay binds sarcosine-1 substituted angiotensins exclusively. Hydrophobic interaction chromatography experiments suggest that these sites, possibly, represent distinct proteins. The third chapter discusses the successful solubilization and partial characterization of the rat brain angiotensin receptor.

  19. Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.

    PubMed Central

    Ballesta, J. J.; Garcia, A. G.; Gutierrez, L. M.; Hidalgo, M. J.; Palmero, M.; Reig, J. A.; Viniegra, S.

    1990-01-01

    1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown. PMID:1704272

  20. Dopamine Inhibits Angiotensin-Stimulated Aldosterone Biosynthesis in Bovine Adrenal Cells

    PubMed Central

    Mc Kenna, Terence J.; Island, Donald P.; Nicholson, Wendell E.; Liddle, Grant W.

    1979-01-01

    The possibility that dopamine may play a role in the in vivo control of aldosterone production in man was suggested to us by reports from others; (a) that bromocriptine, a dopaminergic agonist, inhibits the aldosterone response to diuresis and to the infusion of angiotensin or ACTH; and (b) that metaclopramide, a dopamine blocking agent, causes elevations in plasma aldosterone levels. To determine whether such effects were direct or indirect, we examined the action of dopamine on aldosterone biosynthesis in isolated, bovine adrenal cells. Dopamine significantly inhibits the aldosterone response to angiotensin (P < 0.001), but does not influence basal aldosterone biosynthesis. It has previously been reported that angiotensin stimulates both the early and late phases of aldosterone biosynthesis. The present experiments demonstrated that the enhancing effect of angiotensin on the conversion of deoxycorticosterone to aldosterone (late phase of aldosterone biosynthesis) was almost completely inhibited by dopamine (P < 0.001). A significant inhibitory effect of dopamine (10 nM) was seen even when aldosterone biosynthesis was stimulated by a grossly supraphysiological concentration of angiotensin II (10 μM). However, these studies did not demonstrate any direct effect of dopamine on the early phase of aldosterone biosynthesis (cholesterol to pregnenolone) basally or when stimulated, or on the late phase of aldosterone biosynthesis under basal conditions. These in vitro studies suggest a direct inhibitory role for dopamine on the late phase of aldosterone biosynthesis, which may account for the in vivo inhibition of the aldosterone response to angiotensin in subjects treated with a dopaminergic agent. PMID:447857

  1. Isolation and characterization of a specific endogenous Na/sup +/, K/sup +/-ATPase inhibitor from bovine adrenal

    SciTech Connect

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-06-14

    In order to identify a specific endogenous Na/sup +/,K/sup +/-ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na/sup +/,K/sup +/-ATPase; (ii) competitive displacing activity against (/sup 3/H)ouabain binding to the enzyme; (iii) inhibitory activity for /sup 86/Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C/sub 18/ open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na/sup +/,K/sup +/-ATPase. These results strongly suggest that this water-soluble nonpeptidic Na/sup +/,K/sup +/-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

  2. In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

    PubMed

    Wallat, S; Kunau, W H

    1976-07-01

    Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.

  3. Electrophysiological and morphological features underlying neurotransmission efficacy at the splanchnic nerve-chromaffin cell synapse of bovine adrenal medulla.

    PubMed

    de Diego, Antonio M G

    2010-02-01

    The ability of adrenal chromaffin cells to fast-release catecholamines relies on their capacity to fire action potentials (APs). However, little attention has been paid to the requirements needed to evoke the controlled firing of APs. Few data are available in rodents and none on the bovine chromaffin cell, a model extensively used by researchers. The aim of this work was to clarify this issue. Short puffs of acetylcholine (ACh) were fast perifused to current-clamped chromaffin cells and produced the firing of single APs. Based on the currents generated by such ACh applications and previous literature, current waveforms that efficiently elicited APs at frequencies up to 20 Hz were generated. Complex waveforms were also generated by adding simple waveforms with different delays; these waveforms aimed at modeling the stimulation patterns that a chromaffin cell would conceivably undergo upon strong synaptic stimulation. Cholinergic innervation was assessed using the acetylcholinesterase staining technique on the supposition that the innervation pattern is a determinant of the kind of stimuli chromaffin cells can receive. It is concluded that 1) a reliable method to produce frequency-controlled APs by applying defined current injection waveforms is achieved; 2) the APs thus generated have essentially the same features as those spontaneously emitted by the cell and those elicited by fast-ACh perifusion; 3) the higher frequencies attainable peak at around 30 Hz; and 4) the bovine adrenal medulla shows abundant cholinergic innervation, and chromaffin cells show strong acetylcholinesterase staining, consistent with a tight cholinergic presynaptic control of firing frequency.

  4. Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation.

    PubMed Central

    White, P C; New, M I; Dupont, B

    1984-01-01

    We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450C21). Serum from rabbits immunized with purified P-450C21 precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450C21 on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450C21 was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC X dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450C21 serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti-P-450C21 serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, pC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450C21. The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450C21 and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Images PMID:6609358

  5. A defined, controlled culture system for primary bovine chromaffin progenitors reveals novel biomarkers and modulators.

    PubMed

    Masjkur, Jimmy; Levenfus, Ian; Lange, Sven; Arps-Forker, Carina; Poser, Steve; Qin, Nan; Vukicevic, Vladimir; Chavakis, Triantafyllos; Eisenhofer, Graeme; Bornstein, Stefan R; Ehrhart-Bornstein, Monika; Androutsellis-Theotokis, Andreas

    2014-07-01

    We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.

  6. A Dual-Color Luciferase Assay System Reveals Circadian Resetting of Cultured Fibroblasts by Co-Cultured Adrenal Glands

    PubMed Central

    Noguchi, Takako; Ikeda, Masaaki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

    2012-01-01

    In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture

  7. A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands.

    PubMed

    Noguchi, Takako; Ikeda, Masaaki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

    2012-01-01

    In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture

  8. Fertilizability and developmental capacity of individually cultured bovine oocytes.

    PubMed

    Fukui, Y; Kikuchi, Y; Kondo, H; Mizushima, S

    2000-05-01

    Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.

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

    SciTech Connect

    Tong, W.; Yeung, E.S.

    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}

  10. [Isoenzymatic forms and distribution of adenylate kinase and creatine kinase in the bovine adrenal medulla].

    PubMed

    Miras-Portugal, M T; Orera, A; Millaruelo, A

    1982-01-01

    Adenylate kinase, creatine kinase as well as their substrate and product levels have been investigated in the adrenal medullary tissue. The concentration of adenine nucleotides and creatine + creatine phosphate are 12.6 +/- 0.4 and 6.9 +/- 0.4 mumol/g wet weight respectively. Adenylate kinase is mainly in the cytosol; only 4% was found in mitochondria. The cytosol enzyme presents a Km for AMP of 5 X 10(-4) M and a Ki for diadenosine pentaphosphate of 0.6 X 10(-6) M. In gel electrophoresis, only one band of adenylate kinase activity can be seen, and its mobility is different from that of the brain enzyme. Creatine kinase from adrenal medulla is mainly found in cytosol; only 3-4% was associated with mitochondria. The cytosolic enzyme is mainly the BB isozyme form.

  11. A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques.

    PubMed Central

    Maconochie, D J; Knight, D E

    1992-01-01

    1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600. PMID:1282154

  12. Muscarinic and opioid receptor modulation of release of (Met/sup 5/-enkephalin immunoreactive material and catecholamines from the bovine adrenal gland

    SciTech Connect

    Barron, B.A.

    1985-01-01

    Retrogradely perfused bovine adrenal glands were stimulated by acetylcholine (ACh) and 1,1-dimethyl-4-phenyl-piperazinium (DMPP), with or without: hexamethonium (C-6), atropine, imipramine, methacholine, pilocarpine, etorphine, or diprenorphine. Stimulation by either ACh DMPP resulted in an increased release of both (Met/sup 5/)-enkephalin immunoreactive material (ME-IRM) and catecholamines as measured by radioimmunoassay and high performance liquid chromatography with electrochemical detection, respectively. ACh (5 x 10/sup -5/ M) and DMPP (5 x 10/sup -5/ M) stimulated the release of norepinephrine greater than the release of epinephrine. The action of these agents was antagonized by C-6(5 x 10/sup -4/ M). Atropine (5 x 10/sup -7/ M) antagonized the action of ACh to stimulate norepinephrine and MI-IRM release while having no effect on DMPP-stimulated release. Imipramine (5 x 10/sup -6/ M) had no effect on either ACh or DMPP-stimulated release. Methacholine (4 x 10/sup -5/ M) potentiated the DMPP (1 x 10/sup -5/ M) stimulation of ME-IRM and catecholamine release; pilocarpine (4 x 10/sup -5/ M) significantly potentiated only the DMPP-stimulated release of norepinephrine. Pilocarpine (5 x 10/sup -5/ M) and muscarine (5 x 10/sup -5/ M) had no effect on the secretion of MI-IRM and catecholamines from the bovine adrenal gland. Etorphine (5 x 10/sup -7/ M) significantly decreased the ACh and DMPP stimulation ME-IRM and catecholamine release. The activity of a muscarinic cholinergic receptor in the bovine adrenal medulla in stimulus-secretion coupling has been controversial. The binding of /sup 3/H-quinuclidinyl benzilate to chromaffin granule membranes was investigated to further characterize muscarinic receptors in the bovine adrenal gland.

  13. Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromaffin cells

    PubMed Central

    Engisch, Kathrin L; Nowycky, Martha C

    1998-01-01

    Endocytosis following exocytosis evoked by single step depolarizations was examined in bovine adrenal chromaffin cells using high resolution capacitance measurements in perforated-patch voltage clamp recordings. Endocytosis was detected as a smooth exponential decline in membrane capacitance to either the pre-stimulus level (‘compensatory retrieval’) or far below the pre-stimulus level (‘excess retrieval’). During excess retrieval, > 10 % of the cell surface could be internalized in under 5 s. Compensatory retrieval was equal in magnitude to stimulus-evoked exocytosis for membrane additions > 100 fF (about fifty large dense-cored vesicles). In contrast, excess retrieval surpassed both the stimulus-evoked exocytosis, and the initial capacitance level recorded at the onset of phase-tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly returned to pre-stimulus levels, even in the absence of stimulation. A large percentage of capacitance increases < 100 fF, usually evoked by 40 ms depolarizations, were not accompanied by membrane retrieval. Compensatory retrieval could occur with any amount of Ca2+ entry, but excess retrieval was never triggered below a threshold Ca2+ current integral of 70 pC. The kinetics of compensatory and excess retrieval differed by an order of magnitude. Compensatory retrieval was usually fitted with a single exponential function that had a median time constant of 5.7 s. Excess retrieval usually occurred with double exponential kinetics that had an extremely fast first time constant (median, 670 ms) and a second time constant indistinguishable from that of compensatory retrieval. The speed of compensatory retrieval was Ca2+ dependent: the largest mono-exponential time constants occurred for the smallest amounts of Ca2+ entry and decreased with increasing Ca2+ entry. The Ca2+ dependence of mono-exponential time constants was disrupted by cyclosporin A (CsA), an inhibitor of the Ca2

  14. Bovine lactotroph cultures for the study of prolactin synthesis functions.

    PubMed

    Wang, Jianfa; Yang, Zhanqing; Fu, Shoupeng; Liu, Bingrun; Wu, Dianjun; Wang, Wei; Sun, Dongbo; Wu, Rui; Liu, Juxiong

    2016-03-01

    The aim of this study was to establish a bovine anterior pituitary-derived lactotroph (BAPDL) line that expresses prolactin (PRL) in vitro to study the mechanisms of bovine PRL synthesis and secretion. Immunohistochemistry assay of PRL in the newborn calves' anterior pituitary glands showed that most lactotrophs were located within the superior border of the lateral wings of the anterior pituitary. Tissues of the superior border of the lateral wings of the anterior pituitary were dispersed and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The limiting dilution method was used to establish BAPDL from single cell clone. BAPDL cells constantly expressed mRNAs for PRL and pituitary-specific transcription factor 1 (Pit-1) gene and grew steadily and rapidly in the DMEM supplemented with 10% FBS. PRL immunoreactivity was present in BAPDL at passage 20. The concentration of bovine PRL in BAPDL at passage 20 culture supernatant was decreased to below 35% compared with that in BAPDL at passage 1. The effects of human epidermal growth factor (hEGF) and dopamine (DA) on the expression and secretion of PRL in BAPDL at passage 4 were also investigated. The results are consistent with those of previous studies. Thus, it can be used successfully for studying the mechanisms of stimuli regulating PRL synthesis and release.

  15. Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells.

    PubMed

    Liu, Pei-Shan; Liu, Ging-Hui; Chao, Wei-Liang

    2008-02-03

    Nonylphenol (NP) is the most critical metabolite of alkylphenol polyethoxylate detergents. NP is known as an endocrine disruptor with estrogenic activities and as an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Estrogen has modulatory roles on ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). Ca(2+)-ATPase inhibitors can modulate the cytosolic calcium concentration ([Ca(2+)](c)]) and thus can affect the calcium signaling coupled with nAChRs. Therefore, NP is predicted to have complex effects on the Ca(2+) signaling and secretion coupled with nAChRs. This study investigated these effects using bovine adrenal chromaffin cells. The results show that NP suppressed the Ca(2+) signaling coupled with nAChRs and voltage-operated Ca(2+) channels in a dose-dependent manner, with IC(50)s of 1 and 5.9 microM, respectively. Estradiol exhibits similar suppression but much lower inhibitory potencies. NP alone induced a transient rise in [Ca(2+)](c) in the presence or absence of extracellular calcium. Thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, partially suppressed the [Ca(2+)](c) rise induced by NP, but NP totally blocked the [Ca(2+)](c) rise induced by thapsigargin. This illustrates that NP can cause Ca(2+) release from thapsigargin-insensitive pools. Thapsigargin suppressed the Ca(2+) signaling coupled with nAChRs but increased that coupled with voltage-operated Ca(2+) channels. We propose that three routes are responsible for the effects of NP on nAChRs: named receptor channels, voltage-gated Ca(2+) channels, and Ca(2+)-induced Ca(2+) release. Three routes are related to the characteristics of NP as steroid-like compounds and Ca(2+)-ATPase inhibitor.

  16. Native and recombinant bovine growth hormone antagonize insulin action in cultured bovine adipose tissue.

    PubMed

    Etherton, T D; Evock, C M; Kensinger, R S

    1987-08-01

    The current study was undertaken to determine if pituitary bovine GH (pbGH) and recombinant bGH (rbGH) antagonized insulin action in bovine adipose tissue after acute (2-h) and chronic (48-h) exposure and whether this was an intrinsic property of bGH. Insulin action (measured as the effect on incorporation of acetate-carbon into long-chain fatty acids) was unaffected by bGH in short term incubations regardless of whether hydrocortisone (HC) was present. After 48 h of culture, however, both pbGH and rbGH similarly antagonized the ability of insulin to maintain lipogenic capacity. This antagonism was dependent upon the presence of HC and was dose dependent, with half-maximal inhibition of insulin action occurring at about 0.5 ng/ml bGH. Bovine PRL did not mimic the effects of bGH on insulin action. These results establish that bGH antagonizes insulin action in bovine adipose tissue and that this effect is dependent upon long term exposure and the inclusion of HC in the culture medium. The fact that both rbGH and pbGH acted similarly indicates that this is an intrinsic property of bGH. The effect of bGH on insulin-dependent maintenance of lipogenic capacity may play an important role in redirecting nutrients away from adipose tissue to other tissues, such as muscle or mammary tissue. It is speculated that this metabolic effect of bGH plays an important role in the adaptive response to chronic bGH treatment, which increases milk yield of dairy cows and growth performance of beef cattle.

  17. In vitro development of bovine embryos cultured with activin A.

    PubMed

    Trigal, B; Gómez, E; Díez, C; Caamaño, J N; Martín, D; Carrocera, S; Muñoz, M

    2011-02-01

    The aim of this study was to investigate the effects of activin A on development, differential cell counts and apoptosis/necrosis rates of bovine embryos produced in vitro. Presumptive zygotes were cultured up to Day 8 in synthetic oviduct fluid containing aminoacids, citrate, myo-inositol and BSA. In Experiment 1, activin (10 ng mL(-1)) was added: 1/from Day 1 to Day 3; 2/from Day 1 to Day 8; 3/from Day 3 to Day 8; or 4/absent (control). In Experiment 2, 10 ng mL(-1) activin were added either before (Day 3 to Day 5) or after (Day 5 to Day 8) the early morula stage. In Experiment 1, activin during the first 72 h of culture reduced Day 3 cleavage, 5-8 cell rates and blastocyst development, while hatching rates increased. No changes were observed within differential cell counts. In experiment 2, activin improved blastocyst development after, and had no effect before, the Day 5 morula stage. However, trophectoderm (TE) cell numbers decreased with activin both before and after the Day 5 morula stage, suggesting that activin inhibits TE differentiation. The presence of activin during the whole culture had no effect on TUNEL positive cells, but when added at shorter periods activin increased apoptotic rates. Effects of activin during in vitro bovine embryo development, depends on timing of its addition to the culture medium. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Effects of Cultured Adrenal Chromaffin Cell Implants on Hindlimb Reflexes of the 6-OHDA Lesioned Rat

    PubMed Central

    Pulford, Bruce E.; Mihajlov, Andrea R.; Nornes, Howard O.; Whalen, L. Ray

    1994-01-01

    The effects of implantation of cultured adrenal medullary cells on the recovery of neurotransmitter specific reflex activity were studied in the rat spinal cord using electrophysiological testing methods. Cell suspensions of cultured neonatal adrenal medullary chromaffin (AM) cells (which produce catecholamines), or Schwann (Sc) cells (controls) were implanted into the lumbar region of the spinal cord 2 weeks after catecholamine (CA) denervation by intracisternal injection of 6-hydroxydopamine (6-OHDA). All cells were taken from 7 day neonates and cultured for 10 days in the presence of nerve growth factor (NGF). Three months after implantation, the extent of implant-associated recovery of reflex activity was determined by measuring electromyogram (EMG) activity and force associated with the long latency component of the hindlimb withdrawal reflex (which is CA modulated). After the electrophysiological testing, rats were anesthetized, and the spinal cords were rapidly removed and frozen. Spinal cords were sectioned longitudinally, and implanted cells were visualized using glyoxylic acid techniques. Labelled sections were examined to determine cell survival. Results indicate that 1) chromaffin cells survive for 3 months in the segments of the cord into which they have been implanted and 2) rats implanted with AM cells have significantly more forceful withdrawal reflexes than those that received Sc cells or received no implant after lesioning. PMID:7703294

  19. Antigenicity of Influenza Vaccine from Bovine Cell Cultures

    PubMed Central

    Leiderman, Eduardo; Mogabgab, William J.

    1969-01-01

    An experimental vaccine prepared from influenza virus strains propagated in bovine kidney cell cultures, purified by zonal centrifugation, and further treated with ether was studied in man for the incidence of clinical reactions and hemagglutination-inhibition antibody levels induced. The results were equivalent to those obtained in a simultaneous study made with a commercially licensed influenza vaccine derived from viruses propagated in the embryonated egg and also purified by zonal centrifugation, but not treated with ether. Comparison of the macromethod and the micromethod for determination of hemagglutination-inhibition antibody titers revealed that lower initial titers and lesser increments in antibody levels following vaccination were obtained by the microtechnique. PMID:4905036

  20. Detection of disease-associated prion protein in the optic nerve and the adrenal gland of cattle with bovine spongiform encephalopathy by using highly sensitive immunolabeling procedures.

    PubMed

    Okada, Hiroyuki; Iwamaru, Yoshifumi; Fukuda, Shigeo; Yokoyama, Takashi; Mohri, Shirou

    2012-04-01

    A sensitive immunohistochemical procedure, the tyramide signal amplification (TSA) system, was applied to detect the localization of immunolabeled disease-associated prion protein (PrP(Sc)) in cattle affected with bovine spongiform encephalopathy (BSE). In this procedure, immunolabeling could be visualized in the optic nerve and the adrenal medulla. In the optic nerve, the dual immunofluorescent technique showed that the granular PrP(Sc) was occasionally detected in the astrocytes, microglia, and myelin sheath adjacent to the axon. Clustered PrP(Sc) was also scattered in association with microglial cells and astrocytes of the optic nerve. In the adrenal gland, PrP(Sc) immunolabeling was confined within the sympathetic nerve fibers and endings. The results suggest that (1) PrP(Sc) might centrifugally spread within and between glial cells and/or the non-axonal (also known as ad-axonal) region of nerve fibers, rather than the axonal and/or extracellular space pathway in the optic nerve, and (2) the sympathetic innervations might be important for the trafficking of BSE agent in the adrenal glands of cattle. This study also suggests that tyramide-based immunochemical analysis should be performed to detect immunolabeled PrP(Sc) in the extracerebral tissues of BSE-affected cattle.

  1. Detection of Disease-associated Prion Protein in the Optic Nerve and the Adrenal Gland of Cattle with Bovine Spongiform Encephalopathy by Using Highly Sensitive Immunolabeling Procedures

    PubMed Central

    Iwamaru, Yoshifumi; Fukuda, Shigeo; Yokoyama, Takashi; Mohri, Shirou

    2012-01-01

    A sensitive immunohistochemical procedure, the tyramide signal amplification (TSA) system, was applied to detect the localization of immunolabeled disease-associated prion protein (PrPSc) in cattle affected with bovine spongiform encephalopathy (BSE). In this procedure, immunolabeling could be visualized in the optic nerve and the adrenal medulla. In the optic nerve, the dual immunofluorescent technique showed that the granular PrPSc was occasionally detected in the astrocytes, microglia, and myelin sheath adjacent to the axon. Clustered PrPSc was also scattered in association with microglial cells and astrocytes of the optic nerve. In the adrenal gland, PrPSc immunolabeling was confined within the sympathetic nerve fibers and endings. The results suggest that (1) PrPSc might centrifugally spread within and between glial cells and/or the non-axonal (also known as ad-axonal) region of nerve fibers, rather than the axonal and/or extracellular space pathway in the optic nerve, and (2) the sympathetic innervations might be important for the trafficking of BSE agent in the adrenal glands of cattle. This study also suggests that tyramide-based immunochemical analysis should be performed to detect immunolabeled PrPSc in the extracerebral tissues of BSE-affected cattle. PMID:22260993

  2. Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

    PubMed

    Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

    2010-08-01

    We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

  3. Culturing bovine nucleus pulposus explants by balancing medium osmolarity.

    PubMed

    van Dijk, Bart; Potier, Esther; Ito, Keita

    2011-11-01

    Regenerative therapies are promising treatments for early intervertebral disc degeneration. To test their efficacy, an in vitro tissue-level model would be valuable. Nucleus pulposus (NP) explant culture may constitute such a model, as the earliest signs of degeneration are in the NP. However, in NP explant cultures, balancing tissue osmolarity is crucial to preventing swelling, proteoglycan (PG) loss and, therefore, maintaining a native cell environment. In this study, we investigated the effect of medium osmolarity on NP explants. We hypothesized that balancing the inherent tissue osmolarity would prevent swelling and thus maintain NP tissue in a native state. Bovine NP explants were cultured for 21 days in hypo-, iso-, and hyper-tonic conditions using either sucrose or polyethylene glycol (PEG) to raise medium osmolarity. Explants were analyzed for water and biochemical content, cell viability, gene expression, and tissue histology, and compared to day 0 samples. In hypo-tonic and both sucrose cultures, swelling was not prevented, resulting in PG loss and changes in cell behavior. Only PEG cultures maintained water and biochemical content and a histological aspect similar to those of native tissue, with better results for hyper- than for iso-tonic conditions. Using PEG to raise culture medium osmolarity, we were able to maintain the NP tissue specific matrix composition, important for disc cell behavior. This approach, thus, constitutes a promising model to test regenerative therapies for early intervertebral disc degeneration.

  4. Adrenal cortex carcinomas with distant metastases in beef cattle at slaughter.

    PubMed

    Edwards, J F; Ralston, K E

    2013-07-01

    Ten cases of adrenal cortex carcinomas with distant metastases were collected as subclinical lesions at slaughter of approximately 14,000 adult cattle. The primary lesion in the adrenal gland and the distant metastases, to either the lung or liver, were characterized by light microscopy and immunohistochemistry. Carcinomas were usually detected by noting metastases in the lungs as polypoid, soft, red or red and yellow masses. All adrenal tumours were unilateral and none were seen in bulls. In six of 10 carcinomas there was gross evidence of invasion of the vena cava via the adrenal vein. Normal bovine adrenal cortex labelled positively with S100, calretinin, α inhibin and melan-A; however, adenomas and seven of 10 carcinomas were labelled best by melan-A and α inhibin. Three carcinomas, grossly identical to the other seven, had numerous calcific granules and a slightly different microscopical appearance. In addition to melan-A and α-inhibin, these variant carcinomas labelled with S100. This variant may be derived from a different layer of the adrenal cortex. Because of the similarity of the bovine and human adrenal cortices, cultures of spontaneously arising bovine adrenal tumours may be a useful resource for study of human neoplasia.

  5. Nonreutilizaton of adrenal chromaffin granule membranes following secretion

    SciTech Connect

    Nobiletti, J.B.

    1985-01-01

    The intracellular postexocytotic fate of the adrenal chromaffin granule membrane (reutilization vs. nonreutilization) was addressed through two experimental approaches. First, (/sup 3/H) 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.

  6. Differential effects of heparin on inositol 1,4,5-trisphosphate binding, metabolism, and calcium release activity in the bovine adrenal cortex.

    PubMed

    Guillemette, G; Lamontagne, S; Boulay, G; Mouillac, B

    1989-03-01

    In a wide variety of cells, inositol-1,4,5-triphosphate is a second messenger that interacts with specific intracellular receptors and triggers the release of sequestered Ca2+ from an intracellular store. We have looked at the influence of heparin on the action and metabolism of inositol-1,4,5-triphosphate in the bovine adrenal cortex. Heparin blocked inositol-1,4,5-trisphosphate binding with half-maximal efficiency around 10 micrograms/ml. Scatchard analyses revealed that heparin did not change the affinity but decreased the number of available binding sites. The Ca2+-releasing activity of inositol-1,4,5-trisphosphate was monitored with the fluorescent indicator, fura-2. Heparin blocked this activity with half-maximal effeciency around 10 micrograms/ml. The effect of heparin could be overcome by a supramaximal dose of inositol-1,4,5-trisphosphate (25 microM). The activity of inositol-1,4,5-trisphosphate-3-kinase from bovine adrenal cortex cytosol was also studied. Heparin inhibited the activity of the kinase with a half-maximal effeciency around 0.4 microgram/ml. Lineweaver-Burk plots revealed that this potent effect was noncompetitive. Finally, we observed that heparin is without effect on inositol-1,4,5-trisphosphate-5-phosphatase (at concentrations as high as 2 mg/ml). These results are consistent with the suggestion that the binding sites for inositol-1,4,5-trisphosphate are the intracellular receptors responsible for the Ca2+-mobilizing effects of inositol-1,4,5-trisphosphate. These results also show that the kinase, the phosphatase, and the receptor are three different molecular entities, which are affected in a different manner by heparin.

  7. Inhibition of /sup 22/Na influx by tricyclic and tetracyclic antidepressants and binding of (/sup 3/H)imipramine in bovine adrenal medullary cells

    SciTech Connect

    Arita, M.; Wada, A.; Takara, H.; Izumi, F.

    1987-10-01

    In bovine adrenal medullary cells we investigated the effects of antidepressants on ionic channels and secretion of catecholamines. Tricyclic (imipramine, amitriptyline and nortriptyline) and tetracyclic (maprotiline and mianserin) antidepressants inhibited carbachol-induced influx of /sup 22/Na, /sup 45/Ca and secretion of catecholamines (IC50, 14-96 microM). Influx of /sup 22/Na, /sup 45/Ca and secretion of catecholamines due to veratridine also were inhibited by these drugs (IC50, 10-17 microM). However, antidepressants did not suppress high concentration of K-induced 45Ca influx and catecholamine secretion, suggesting that antidepressants do not inhibit voltage-dependent Ca channels. (/sup 3/H)Imipramine bound specifically to adrenal medullary cells. Binding was saturable, reversible and with two different equilibrium dissociation constants (13.3 and 165.0 microM). Tricyclic and tetracyclic antidepressants competed for the specific binding of (/sup 3/H)imipramine at the same concentrations as they inhibited /sup 22/Na influx caused by carbachol or veratridine. Carbachol, d-tubocurarine, hexamethonium, tetrodotoxin, veratridine and scorpion venom did not inhibit the specific binding of (/sup 3/H)imipramine. These results suggest that tricyclic and tetracyclic antidepressants bind to two populations of binding sites which are functionally associated with nicotinic receptor-associated ionic channels and with voltage-dependent Na channels, and inhibit Na influx. Inhibition of Na influx leads to the reduction of Ca influx and catecholamine secretion caused by carbachol or veratridine.

  8. The minotaur proteome: avoiding cross-species identifications deriving from bovine serum in cell culture models.

    PubMed

    Bunkenborg, Jakob; García, Guadalupe Espadas; Paz, Marcia Ivonne Peña; Andersen, Jens S; Molina, Henrik

    2010-08-01

    Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5-15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented.

  9. Hypertrophy of cultured bovine aortic endothelium following irradiation

    SciTech Connect

    Rosen, E.M.; Vinter, D.W.; Goldberg, I.D.

    1989-03-01

    The vascular endothelium is a vital multifunctional tissue which covers the entire luminal surface of the circulatory system. Loss of continuity of the endothelial lining normally results in cell migration and proliferation to make up for cell loss and to ensure that exposure of the thrombogenic subendothelium to platelets and clotting factors is minimized. We showed that ionizing radiation (400-3000 cGy) causes dose-dependent cell loss from confluent monolayer cultures of bovine aortic endothelium, which cannot immediately be compensated by cell proliferation. Within 24 h, the remaining attached cells undergo substantial somatic hypertrophy (evidenced by increased protein content, cell volume, and attachment area) but remain diploid. If cell loss is not excessive, monolayer continuity is restored within several days. Although reduced protein degradation may contribute, most of the protein accumulation is due to synthesis of new protein. Unlike endothelium, irradiation of smooth muscle cultures causes neither cell loss nor increased protein synthesis. Hypertrophy of irradiated endothelial cells appears to be a consequence of a proliferative stimulus (cell loss) in a population of cells which is unable to divide. It can be modulated by replating irradiated cells at different densities. We suggest that endothelial hypertrophy is an early vascular homeostatic response before clonal proliferation of surviving cells or repopulation by cells from outside of the irradiated field can compensate for cell loss.

  10. Proteomic analysis of Escherichia coli O157 cultured in bovine rumen fluid

    USDA-ARS?s Scientific Manuscript database

    To obtain insights into Escherichia coli O157 (O157) adaptation and survival in the bovine rumen, the first anatomical compartment encountered by this pathogen during transit through the bovine gastrointestinal tract to sites of colonization, we defined the proteome of O157 cultured in rumen fluid (...

  11. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

    PubMed

    Föllmann, W; Weber, S; Birkner, S

    2000-10-01

    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  12. Ethanolamine metabolism in cultured bovine aortic endothelial cells

    SciTech Connect

    Lipton, B.A.; Davidson, E.P.; Ginsberg, B.H.; Yorek, M.A. )

    1990-05-05

    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.

  13. Mas-related G-protein-coupled receptor c agonist bovine adrenal medulla 8-22 attenuates bone cancer pain in mice

    PubMed Central

    Sun, Yu-E; Lu, Cui-E; Lei, Yishan; Liu, Yue; Ma, Zhengliang; Gu, Xiaoping

    2015-01-01

    Objectives: The aim of this study is to investigate the effects of Mas-related G-protein-coupled receptor C (MrgC) agonist bovine adrenal medulla 8-22 (BAM8-22) on bone cancer pain and mirror-image pain. Methods: Bone cancer pain was induced by intramedullary injection of NC2472 fibrosarcoma cells in the mice. BAM8-22 and/or anti-MrgC antibody were injected intrathecally at day 14 after bone cancer induction and their effects on pain behaviors were detected. The pain behaviours were assessed by the number of spontaneous foot lifts and paw withdrawal mechanical threshold (PWMT) tests. MrgC expression was detected using western blot analysis and immunofluorescence assay. Results: There were increased bone cancer pain and mirror-image pain in the tumor-bearing mice while not in the sham-treated mice. BAM8-22 attenuated bone cancer pain in mice dose dependently with the highest effects at 2 hr after BAM8-22 administration, and anti-MrgC antibody reversed the effects of BAM8-22. However, intrathecal administration of BAM8-22 did not affect the mirror-image pain. Furthermore, BAM8-22 stimulated the expression of MrgC in the spinal dorsal horn. Conclusions: MrgC agonist BAM8-22 could attenuate bone cancer pain in mice. This study may provide a novel strategy for the treatment of bone cancer pain. PMID:26884930

  14. [Characteristics of steroid metabolism in transgenic Nicotiana tabacum plants bearing the CYP11A1 cDNA of cytochrome P450(SCC) from the bovine adrenal cortex].

    PubMed

    Spivak, S G; Berdichevets, I N; Litvinovskaia, R P; Drach, S V; Kartel', N A; Shpakovskiĭ, G V

    2010-01-01

    In the mitochondria of animal steroidogenic tissues, cytochrome P450(SCC), encoded by the CYP11A1 gene, catalyzes the conversion of cholesterol into pregnenolone - the general precursor of all steroid hormones. In this work, we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA cytochrome P450(SCC)from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wild-type plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone - the product of a reaction catalyzed by cytochrome P450(SCC). The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type. The seeds of the transgenic plants contain less (24R)-brassinosteroids than the wild-type tobacco plants. The results obtained indicate that the synthesis of an active P450(SCC) cytochrome in transgenic Nicotiana tabacum plants has a profound effect on steroid metabolism and is responsible for the specific phenotypic features of transgenic plants bearing CYP11A1 cDNA.

  15. Generation of large pig and bovine blastocysts by culturing in human induced pluripotent stem cell medium.

    PubMed

    Gao, Qing-Shan; Jin, Long; Li, Suo; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Jin, Qing-Guo; Kang, Jin-Dan; Yan, Chang-Guo; Yin, Xi-Jun

    2016-04-01

    We investigated the effect of human induced pluripotent stem cell (hiPS) medium on porcine somatic cell nuclear transfer and bovine in vitro fertilized early blastocysts, in comparison with North Carolina State University (NCSU)-37 medium and in vitro culture (IVC)-II medium. After 2 days of culture, the diameter of the portion of the blastocyst that was extruded from the zona pellucid dramatically differed between porcine blastocysts cultured in hiPS medium and those cultured in NCSU-37 medium (221.47 ± 38.94 μm versus 481.87 ± 40.61 μm, P < 0.01). Moreover, the diameter of the portion of the blastocyst significantly differed between bovine blastocysts cultured in hiPS medium and those cultured in IVC-II medium (150.30 ± 29.49 μm versus 195.58 ± 41.59 μm, P < 0.01). Furthermore, the total number of cells per porcine and bovine blastocyst was more than two-fold higher in blastocysts cultured in hiPS medium than in those cultured in NCSU-37 medium (44.33 ± 5.28 and 143.33 ± 16.05, P < 0.01) or IVC-II medium (172.12 ± 45.08 and 604.83 ± 242.64, P < 0.01), respectively. These results indicate that hiPS medium markedly improves the quality of porcine and bovine blastocysts.

  16. Alkaline buffers release EDRF from bovine cultured aortic endothelial cells.

    PubMed Central

    Mitchell, J. A.; de Nucci, G.; Warner, T. D.; Vane, J. R.

    1991-01-01

    1. Release of endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2) from bovine cultured aortic endothelial cells (EC) was measured by bioassay and radioimmunoassay, respectively. 2. Bradykinin (BK, 3-30 pmol), adenosine diphosphate (ADP, 2-6 nmol) or the sodium ionophore monensin (40-100 nmol) injected through a column of EC released EDRF. L-Arginine free base (FB; 10-20 mumol) or D-arginine FB (10-20 mumol) injected through the column of EC released similar amounts of EDRF and also caused an increase in pH of the Krebs solution perfusing the EC from 7.5-8.0 to 8.6-9.5. Sodium carbonate (Na2CO3) an alkaline buffer which caused the same changes in the pH of the Krebs solution also induced the same release of EDRF. The hydrochloride salts of L- or D-arginine did not cause either release of EDRF when injected through the column of EC or increases in the pH of the Krebs solution. 3. Inhibitors of either diacylglycerol lipase (RHC 80267) or kinase (R59022) inhibited the release of EDRF induced by BK or ADP but potentiated the release induced by L-arginine FB, monensin (40-100 nmol) or alkaline buffer (Na2CO3). R59022 and RHC 80267 infused through the EC increased the basal release of EDRF. 4. When calcium chloride was omitted from the Krebs solution the release of EDRF induced by alkaline buffer (Na2CO3; pH 8.6-9.5) or L-arginine FB (10-20 mumol) was selectively inhibited when compared to that induced by BK (3-30 pmol) or ADP (2-6 nmol). This inhibition was reversed when calcium (2.5 mM) was restored. 5. NG-monomethyl-L-arginine (NMMA; 30 microM) inhibited release of EDRF induced by BK (10-30 pmol) or alkaline buffers (Na2CO3 or D-arginine FB; pH 8.6-9.5). This inhibition was partially reversed by L- but not D-arginine FB or HCl (30-100 microM). 6. Prostacyclin was released when BK (10 pmol), ADP (2 nmol) or arachidonic acid (30 nmol) were injected through the column of EC. However, monensin (40 nmol) or alkaline buffers (pH 8.6-9.5) did not release

  17. Mouse Adrenal Chromaffin Cell Isolation

    PubMed Central

    Kolski-Andreaco, Aaron; Cai, Haijiang; Currle, D. Spencer; Chandy, K. George; Chow, Robert H.

    2007-01-01

    Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated. PMID:18830430

  18. Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

    SciTech Connect

    De Lean, A.; Cantin, M.

    1986-03-05

    The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levels of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with (/sup 32/P) NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production.

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

    PubMed

    Vilela, Ricardo Chaves; Benchimol, Marlene

    2013-02-01

    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.

  20. Properties of soluble and membrane bound dopamine-beta-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate.

    PubMed

    Miras-Portugal, M T; Millaruelo, A; Vara, F

    1980-12-10

    Bovine dopamine-beta-monooxygenase from chromaffin granules in its soluble and membrane-bound forms was cross-linked with the bifunctional reagent dimethyl suberimidate, and its structural and kinetic properties were studied. 1. The cross-linking reaction does not affect the activity of soluble dopamine-beta-monooxygenase; it produces a ten percent inactivation in the membrane-bound enzyme, possibly because the linkage to other membrane proteins hinders its activity. 2. The soluble dopamine-beta-monooxygenase reaction mixture was analyzed by sodium dodecyl sulfate gel electrophoresis, showing appreciable amounts of dimer and tetramer, but only small amounts of trimer. In membrane-bound dopamine-beta-monooxygenase, subjected to the same treatment, appreciable amounts of dimer and higher aggregates were found. 3. The kinetic properties of soluble dopamine-beta-monooxygenase after the crosslinking reaction are the same as those of the native enzyme, with a ping-pong kinetic mechanism and the same real Michaelis constants for tyramine and ascorbate: KmT = 0.36 mM and KmA = 0.32 mM. Membrane-bound dopamine-beta-monooxygenase does not present a ping-pong mechanism before or after cross-linking; its real Michaelis constants are slightly modified by the cross-linking reaction: KmT = 0.4 mM and KMA = 0.4 mM.

  1. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  2. The Effect of Culture Methods and Serum Supplementation on Developmental Competence of Bovine Embryos Cultured In Vitro

    USDA-ARS?s Scientific Manuscript database

    The objective of this study was to compare the developmental competence of bovine in vitro fertilized embryos in three different culture methods; microdrop method (50 µl of medium under mineral oil in petri dishes) compared to tube methods (1 ml of medium in tubes) with or without oil overlay, and t...

  3. Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct.

    PubMed

    Rizos, D; Ramirez, M A; Pintado, B; Lonergan, P; Gutierrez-Adan, A

    2010-04-01

    The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model.

  4. Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos.

    PubMed

    Batista, M; Torres, A; Diniz, P; Mateus, L; Lopes-da-Costa, L

    2012-10-01

    The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days 2-8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for co-culture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P(4)) were evaluated. The production of P(4) was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P(4), PGF(2α), and PGE(2) compared to fresh cell cultures. This enables the use of pools of frozen-thawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P(4) quantified in the medium, but had no effect on PGF(2α) and PGE(2) quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed.

  5. Adrenal glands

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/002219.htm Adrenal glands To use the sharing features on this page, please enable JavaScript. The adrenal glands are two triangle-shaped glands. One gland is ...

  6. Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems.

    PubMed

    Somfai, Tamás; Inaba, Yasushi; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Shuji; Konishi, Kazuyuki; Nagai, Takashi; Imai, Kei

    2010-12-01

    The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.

  7. Characterization of bovine embryos cultured under conditions appropriate for sustaining human naïve pluripotency

    PubMed Central

    van Tol, Helena T. A.; Groot Koerkamp, Marian J. A.; Wubbolts, Richard W.; Haagsman, Henk P.; Roelen, Bernard A. J.

    2017-01-01

    In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a bovine pluripotent cell population, nor how to maintain these cells in vitro. The objective of this study was to determine the transcriptomic profile related to bovine pluripotency. Therefore, in vitro derived embryos were cultured in various culture media that recently have been reported capable of maintaining the naïve pluripotent state of human embryonic cells. Gene expression profiles of embryos cultured in these media were compared using microarray analysis and quantitative RT-PCR. Compared to standard culture conditions, embryo culture in ‘naïve’ media reduced mRNA expression levels of the key pluripotency markers NANOG and POU5F1. A relatively high percentage of genes with differential expression levels were located on the X-chromosome. In addition, reduced XIST expression was detected in embryos cultured in naïve media and female embryos contained fewer cells with H3K27me3 foci, indicating a delay in X-chromosome inactivation. Whole embryos cultured in one of the media, 5iLA, could be maintained until 23 days post fertilization. Together these data indicate that ‘naïve’ conditions do not lead to altered expression of known genes involved in pluripotency. Interestingly, X-chromosome inactivation and development of bovine embryos were dependent on the culture conditions. PMID:28241084

  8. Adrenal insufficiency.

    PubMed

    Auron, Moises; Raissouni, Nouhad

    2015-03-01

    Adrenal insufficiency is a life-threatening condition that occurs secondary to impaired secretion of adrenal glucocorticoid and mineralocorticoid hormones. This condition can be caused by primary destruction or dysfunction of the adrenal glands or impairment of the hypothalamic-pituitary-adrenal axis. In children, the most common causes of primary adrenal insufficiency are impaired adrenal steroidogenesis (congenital adrenal hyperplasia) and adrenal destruction or dysfunction (autoimmune polyendocrine syndrome and adrenoleukodystrophy), whereas exogenous corticosteroid therapy withdrawal or poor adherence to scheduled corticosteroid dosing with long-standing treatment constitute the most common cause of acquired adrenal insufficiency. Although there are classic clinical signs (eg, fatigue, orthostatic hypotension, hyperpigmentation, hyponatremia, hyperkalemia, and hypoglycemia) of adrenal insufficiency, its early clinical presentation is most commonly vague and undefined, requiring a high index of suspicion. The relevance of early identification of adrenal insufficiency is to avoid the potential lethal outcome secondary to severe cardiovascular and hemodynamic insufficiency. The clinician must be aware of the need for increased corticosteroid dose supplementation during stress periods.

  9. [In vitro fertilization of bovine oocytes in in-vitro protein-free culture system].

    PubMed

    Smetanina, I G; Tatarinova, L V; Krivokharchenko, A S

    2006-01-01

    We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.

  10. Increased TH-thymidine incorporation into DNA of organ-cultured adrenal explants from rats injected with corticotropin and/or cysteamine

    SciTech Connect

    Sewerynek, E.; Szkudlinski, M.; Lewinski, A.; Kunert-Radek, J.

    1988-11-30

    The effect of a single injection of cysteamine /CySH/ - a sulfhydryl substance, known to deplete tissue content of somatostatin /SS/ - on TH-thymidine incorporation into DNA of rat adrenal explants incubated in vitro was investigated. It was shown that: 1/ Single in vivo injection of ACTH or of CySH increased TH-thymidine incorporation into DNA of the organ-cultured adrenals, 2/ Dexamethasone reduced the TH-thymidine uptake, but that decrease did not attain statistical significance versus controls.

  11. Identification of bovine embryos cultured in groups by attachment of barcodes to the zona pellucida.

    PubMed

    Novo, Sergi; Morató, Roser; Penon, Oriol; Duran, Sara; Barrios, Leonardo; Nogués, Carme; Plaza, José Antonio; Pérez-García, Luisa; Mogas, Teresa; Ibáñez, Elena

    2014-06-01

    The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each individual female have to be cultured individually or in very small groups. However, it has been demonstrated that single-embryo culture is less efficient than embryo culture in groups. To overcome this limitation, we developed a direct embryo-tagging system, which allows the collective culture of embryos from different origins whilst preserving their pedigree. Presumptive bovine zygotes were tagged with eight wheat-germ agglutinin biofunctionalised polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Four different barcodes were used to encode groups of 20-25 embryos, which were then cultured in the same drop. Cleavage, Day-7 and Day-8 blastocysts and barcode retention rates were assessed. In addition, Day-7 blastocysts were vitrified and warmed. Barcode attachment to the ZP of bovine embryos affected neither in vitro embryo development nor post-warming survival of the tagged embryos. All the embryos maintained barcodes attached until Day 8 of culture (3.63±0.37 barcodes per embryo) and could be identified. In conclusion, identification of embryos by barcodes attached to the ZP is feasible and will allow the culture of embryos from different donors in the same drop.

  12. Cerebellin in the rat adrenal gland: gene expression and effects of CER and [des-Ser1]CER on the secretion and growth of cultured adrenocortical cells.

    PubMed

    Rucinski, Marcin; Albertin, Giovanna; Spinazzi, Raffaella; Ziolkowska, Agnieszka; Nussdorfer, Gastone G; Malendowicz, Ludwick K

    2005-03-01

    Cerebellin (CER) is a regulatory peptide, originally isolated from rat cerebellum, which derives from the cleavage of precerebellin (Cbln), three types of which (Cbln1-3) have been identified in humans and rats. CER is also expressed in several extra-cerebellar tissues, including adrenal gland, and evidence has been provided that CER exerts a modulatory action on human and rat adrenal gland. Hence, we have investigated the expression of Cbln1-3 mRNAs and CER protein-immunoreactivity (IR) in the various zones of rat adrenal glands, and the effects of CER and its metabolite [des-Ser(1)]CER (des-CER) on the secretion and growth of cultured rat adrenocortical cells. Reverse transcription-polymerase chain reaction showed high and low expression of Cbln2 mRNA in zona glomerulosa (ZG) and zona fasciculata-reticularis, respectively. Cbln1 was not expressed, and Cbln3 mRNA was detected only in ZG. No Cbln expression was found in adrenal medulla. Immunocytochemistry demonstrated the presence of CER-IR exclusively in the adrenal cortex, the reaction being more intense in ZG. As expected, ACTH (10(-8) M) markedly enhanced corticosterone secretion and lowered proliferation rate of cultured adrenocortical cells. CER was ineffective, while des-CER exerted an ACTH-like effect, but only at the lowest concentration (10(-10) M). Taken together, these findings allow us to conclude that CER is expressed in rat adrenal cortex, and to suggest that CER conversion to des-CER by endopeptidases is needed for CER to exert its autocrine-paracrine regulatory functions.

  13. Development of a low-serum medium for the production of monoclonal antibody against congenital adrenal hyperplasia by hybridoma culture.

    PubMed

    Chua, Gek Kee

    2016-10-02

    Statistically designed experiments were used in developing a low-serum medium for the production of a diagnostic monoclonal antibody against congenital adrenal hyperplasia using hybridoma 192. A two-level half-fractional factorial design was used for screening six components (Minimum Essential Medium Eagle amino acids, 2-mercaptoethanol, ethanolamine, ferric citrate, zinc sulfate, and sodium selenite). The experimental design was then augmented to central composite design. The basal Dulbecco's modified Eagle's medium (DMEM; containing 4 mM L-glutamine, 1% antibiotic-antimycotic agent) supplemented with 0.4% by volume fetal bovine serum (FBS), 311.8 mM ferric citrate, 17.3 nM sodium selenite, and 4.5 mM zinc sulfate (LSD) was found to support the growth of the hybridoma. Specific cell growth rate in the LSD (0.033 ± 0.001/h) was slightly lower than in the control medium (i.e., basal DMEM supplemented with 2% FBS; 0.0045 ± 0.003/h). Nevertheless, the specific MAb production rate for LSD was higher (0.057 ± 0.015 pg/cell · h versus 0.004 ± 0.002 pg/cell · h in LSD and control, respectively). The antibody produced in the LSD showed high specificity and no cross-reactivity with the other structural resemblance's steroid hormones, revealing no structural changes owing to the new medium formulation developed. The new medium formulation effectively reduced the medium cost by up to 64.6%.

  14. Foetal bovine serum-derived exosomes affect yield and phenotype of human cardiac progenitor cell culture

    PubMed Central

    Angelini, Francesco; Ionta, Vittoria; Rossi, Fabrizio; Miraldi, Fabio; Messina, Elisa; Giacomello, Alessandro

    2016-01-01

    Introduction: Cardiac progenitor cells (CPCs) represent a powerful tool in cardiac regenerative medicine. Pre-clinical studies suggest that most of the beneficial effects promoted by the injected cells are due to their paracrine activity exerted on endogenous cells and tissue. Exosomes are candidate mediators of this paracrine effects. According to their potential, many researchers have focused on characterizing exosomes derived from specific cell types, but, up until now, only few studies have analyzed the possible in vitro effects of bovine serum-derived exosomes on cell proliferation or differentiation. Methods: The aim of this study was to analyse, from a qualitative and quantitative point of view, the in vitro effects of bovine serum exosomes on human CPCs cultured either as cardiospheres or as monolayers of cardiosphere-forming cells. Results: Effects on proliferation, yield and molecular patterning were detected. We show, for the first time, that exogenous bovine exosomes support the proliferation and migration of human cardiosphere-forming cells, and that their depletion affects cardiospheres formation, in terms of size, yield and extra-cellular matrix production. Conclusion: These results stress the importance of considering differential biological effects of exogenous cell culture supplements on the final phenotype of primary human cell cultures. PMID:27340620

  15. Effects of timolol on bovine corneal endothelial cultures.

    PubMed

    Staatz, W D; Radius, R L; Van Horn, D L; Schultz, R O

    1981-04-01

    The chronic use of timolol (Timoptic) maleate to control glaucoma may produce cytotoxic complications in the cornea. We have therefore compared the relative toxic effects of the commercial ophthalmic preparation with that of the pure compound. Commercial vehicle, either with or without 16 mM timolol maleate, killed cultures within the first five minutes of exposure. Pure timolol maleate, however, caused rapid but reversible cellular contractions, and cells remained viable in it for over 24 hours. Dilution with culture medium reduced both the cytotoxicity and the speed of the contractions. Incubation in 1:100 dilutions of vehicle or commercial drug preparations or in 0.16 mM pure timolol maleate did not alter cellular morphology. The results indicate that while undiluted vehicle is toxic, timolol maleate is not.

  16. Effects of IGF-1 on In Vitro Culture of Bovine Preantral Follicles are Dose-Dependent.

    PubMed

    Jimenez, C R; de Azevedo, J L; Silveira, R G; Penitente-Filho, J; Carrascal-Triana, E L; Zolini, A M; Araujo, V R; Torres, Caa; Gonçalves, W G

    2016-06-01

    This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF-1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α-MEM(+) , supplemented with different concentrations of human recombinant IGF-1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO2 in 24-well plates with total replacement of the medium every 2 days. Non-cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF-1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF-1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF-1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF-1 tested. Ultrastructurally, the non-cultured control and IGF-1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF-1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.

  17. The global effect of heat on gene expression in cultured bovine mammary epithelial cells.

    PubMed

    Li, Lian; Sun, Yu; Wu, Jie; Li, Xiaojuan; Luo, Man; Wang, Genlin

    2015-03-01

    Heat stress (HS) in hot climates is a major cause that strongly negatively affects milk yield in dairy cattle, leading to immeasurable economic loss. The heat stress response of bovine mammary epithelial cells (BMECs) is one component of the acute systemic response to HS. Gene networks of BMECs respond to environmental heat loads with both intra- and extracellular signals that coordinate cellular and whole-animal metabolism. Our experimental objective was to characterize the direct effects of heat stress on the cultured bovine mammary epithelial cells by microarray analyses. The data identified 2716 differentially expressed genes in 43,000 transcripts which were changed significantly between heat-stressed and normal bovine mammary epithelial cells (fold change ≥2, P ≤ 0.001). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these differentially expressed genes are involved in different pathways that regulate cytoskeleton, cell cycle, and stress response processes. Our study provides an overview of gene expression profile and the interaction between gene expression and heat stress, which will lead to further understanding of the potential effects of heat stress on bovine mammary glands.

  18. Morphological and functional characterization of bovine oviductal epithelial cell monolayers cultured on polarizing membranes.

    PubMed

    Gómez, E; Uría, H

    1997-01-01

    Several characteristics of oviductal cells, cultured under either polarizing or nonpolarizing conditions, were studied. In vitro produced bovine embryos tested the embryotrophic abilities of the respective conditioned media. Conditioned medium from the apical face of polarized cell monolayers supported higher rates of development to blastocyst and expanded blastocysts. In contrast, conditioned medium from the basal face supported embryo development only to the 8-16 cell stage; however, these embryos were able to continue development to the morula stage when cultured in medium from the apical and basal faces, indicating total cell confluence and a clear functional polarization. At the ultrastructural level, cells cultured in polarizing conditions displayed characteristics nearer to the same cells in vivo and signs of a metabolic activity higher than that in cells cultured under non-polarizing conditions. It can be concluded that cell-polarization, in our culture conditions, is beneficial to embryo development.

  19. Adrenal autotransplantation.

    PubMed

    Srougi, M; Gittes, R F

    1978-04-01

    New tools for the diagnosis of adrenal diseases and the development of successful techniques to treat patients with bilateral tumors of the kidney have increased the number of procedures involving removal of both adrenals. Offering to these patients an adrenal autograft represents more than a superfluous medical exercise, since a successful outcome of the graft will relieve them of the burdens and risks of long-term postoperative steroid replacement therapy. The aim of this review is to bring to mind the possibility of autografting adrenal glands in some clinical situations and to emphasize some points that could be relevant in obtaining successful results. The available data justify clinical trials with the procedure.

  20. Effect of sericin on preimplantation development of bovine embryos cultured individually.

    PubMed

    Isobe, T; Ikebata, Y; Onitsuka, T; Wittayarat, M; Sato, Y; Taniguchi, M; Otoi, T

    2012-09-01

    The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.

  1. Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality.

    PubMed

    Lonergan, P; Rizos, D; Kanka, J; Nemcova, L; Mbaye, A M; Kingston, M; Wade, M; Duffy, P; Boland, M P

    2003-09-01

    The aim of this study was to examine the temporal sensitivity of bovine embryos to culture environment after fertilization to determine which period, if any, is most critical in determining blastocyst quality. Bovine zygotes produced in vitro were divided into six groups and cultured either in vitro (in synthetic oviductal fluid, SOF), in vivo (in the ewe oviduct) or in a combination of both systems. Development to the blastocyst stage, the ability of the blastocysts to withstand cryopreservation and the relative abundance of several gene transcripts were examined. Culture in SOF for either 2 or 4 days, followed by subsequent culture in the ewe oviduct, resulted in a significantly lower yield of blastocysts than did all other methods, the effect being most marked in embryos that were cultured in SOF for 4 days. In contrast, culture in vivo for the first 2 or 4 days after fertilization followed by culture in vitro did not have such a marked effect on blastocyst development. Blastocysts produced after culture in the oviduct for 6 days had the highest rates of survival over 72 h after warming (100% survival at 24 h; >95% survival at 72 h). The embryos that spent the last 4 days of culture in vivo also had relatively high rates of survival (100% at 24 h, 73.7% at 72 h). Blastocysts produced entirely in SOF had very low rates of survival after vitrification, with <40% viable at 24 h and <20% survival at 72 h. Blastocysts derived from embryos that spent the first 2 days in vivo and the last 4 days in vitro had the lowest rates of survival (6.7%), whereas those that spent the last 2 days only in SOF had intermediate rates of survival (40.6%). These differences were reflected in the relative abundance of transcripts for the Bax gene.

  2. Growth and antrum formation of bovine primary follicles in long-term culture in vitro.

    PubMed

    Sun, Jing; Li, Xiangdong

    2013-09-01

    Successful antral formation in vitro from bovine preantral follicles (145-170 μm) has been described previously, but antrum formation from the primary follicle (50-70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50-70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E2 + bFGF or FSH + LH + E2 + bFGF + EGF (p<0.05). An addition of 50 ng/ml bFGF or bFGF +25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF +25 ng/ml EGF to media containing FSH + LH + E2 turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.

  3. Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.

    PubMed

    Ohboshi, S; Etoh, T; Sakamoto, K; Fujihara, N; Yoshida, T; Tomogane, H

    1997-04-15

    Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.

  4. Adrenal Insufficiency

    MedlinePlus

    ... three types of steroid hormones. In adrenal insufficiency (AI), the cortex does not make enough steroid hormones. ... unlike “adrenal fatigue.” There are two kinds of AI: • Primary AI, also called Addison’s disease. In this ...

  5. Three-dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells.

    PubMed

    Zhan, Kang; Lin, Miao; Liu, MingMei; Sui, YangNan; Babekir, Haitham Mohammed; Zhao, GuoQi

    2017-05-01

    Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro. © 2016 Japanese Society of Animal Science.

  6. Bovine whole-blood culture as a tool for the measurement of endotoxin activities in Gram-negative bacterial vaccines.

    PubMed

    Imamura, Saiki; Nakamizo, Mari; Kawanishi, Michiko; Nakajima, Nao; Yamamoto, Kinya; Uchiyama, Mariko; Hirano, Fumiya; Nagai, Hidetaka; Kijima, Mayumi; Ikebuchi, Ryoyo; Mekata, Hirohisa; Murata, Shiro; Konnai, Satoru; Ohashi, Kazuhiko

    2013-05-15

    In order to analyze bovine immune reactions against the Gram-negative bacterial vaccine, bovine whole-blood culture was used to investigate the pro-inflammatory cytokine responses stimulated with lipopolysaccharides (LPS) extracted from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Klebsiella pneumoniae. We also examined the interaction between LPS and aluminum hydroxide gel for endotoxin activity and pro-inflammatory cytokine responses of whole bovine blood. Alteration in the mRNA concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10 in whole-blood culture at 4h after stimulation with different doses of LPS was observed and determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The mRNA concentrations of TNF-α and IL-1β changed in a dose-dependent manner and differed depending on the type of LPS. Limulus test revealed that endotoxin activity was remarkably reduced when aluminum hydroxide gel was added to LPS. In contrast, the mRNA concentration of TNF-α in whole bovine blood was enhanced by LPS mixed with aluminum hydroxide gel. These results suggest that bovine whole-blood culture can be utilized to detect endotoxin activity of Gram-negative bacterial vaccines. In addition, whole-blood culture offers several advantages, such as ease of performance, few preparation artifacts, and a physiological cell environment, for investigating bovine immune response compared with the Limulus test.

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

    PubMed Central

    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

  8. Evaluation of different culture systems on the in vitro production of bovine embryos.

    PubMed

    Pereira, Daniela Costa; Dode, Margot Alves Nunes; Rumpf, Rodolfo

    2005-03-01

    The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified "well of the well" (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar (P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower (P < 0.05) in embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar (P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates (P < 0.05) were observed in the T3 group. In conclusion, embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system.

  9. The control of steroidogenesis by human fetal adrenal cells in tissue culture. IV. The effect of exposure to placental steroids.

    PubMed

    Fujieda, K; Faiman, C; Feyes, F I; Winter, J S

    1982-01-01

    The effect upon steroidogenesis of adding various steroids produced by the placenta was studied in short term cultures of human fetal adrenal cells. The addition of high concentrations (10(3) ng/ml) of estrone or estriol inhibited the production of cortisol, but only the former elicited a parallel increase in dehydroepiandrosterone (DHA) production. Estradiol was effective in inhibiting delta-4-3-ketosteroid production at concentrations of 10-100 ng/ml, levels which approach those found in the fetal circulation, while DHA production was increased at concentrations of 1 microgram/ml. The addition of progesterone (4 microgram/ml) to the medium caused increased production of cortisol and corticosterone, but had no effect on DHA production. Pregnenolone (4 microgram/ml) increased the basal production of DHA and slightly impaired both basal and ACTH-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect-stimulated aldosterone production, but had no effect on cortisol production. The data demonstrate that the many fetal and placental factors which have been studied to date, only ACTH and estrogens can interact to produce the characteristic fetal pattern of steroidogenesis. Preliminary studies indicate that this effect of estrogen is not influenced by other peptide hormones such as hCG, human prl, beta-lipotropin, corticotropin-like intermediate lobe peptide, or beta-endorphin. A revised model of

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

    ERIC Educational Resources Information Center

    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…

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

    ERIC Educational Resources Information Center

    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…

  12. Enhanced BDNF signalling following chronic hypoxia potentiates catecholamine release from cultured rat adrenal chromaffin cells

    PubMed Central

    Scott, Angela L; Zhang, Min; Nurse, Colin A

    2015-01-01

    Environmental stressors, including chronic hypoxia, enhance the ability of adrenomedullary chromaffin cells (AMCs) to secrete catecholamines; however, the underlying molecular mechanisms remain unclear. Here, we investigated the role of brain-derived neurotrophic factor (BDNF) signalling in rat AMCs exposed to chronic hypoxia. In rat adrenal glands, BDNF and its tropomyosin-related kinase B (TrkB) receptor are highly expressed in the cortex and medulla, respectively. Exposure of AMCs to chronic hypoxia (2% O2; 48 h) in vitro caused a significant increase to TrkB mRNA expression. A similar increase was observed in an immortalized chromaffin cell line (MAH cells); however, it was absent in MAH cells deficient in the transcription factor HIF-2α. A specific TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), stimulated quantal catecholamine secretion from chronically hypoxic (CHox; 2% O2) AMCs to a greater extent than normoxic (Nox; 21% O2) controls. Activation of TrkB by BDNF or 7,8-DHF increased intracellular Ca2+ ([Ca2+]i), an effect that was significantly larger in CHox cells. The 7,8-DHF-induced [Ca2+]i rise was sensitive to the tyrosine kinase inhibitor K252a and nickel (2 mm), but not the Ca2+ store-depleting agent cyclopiazonic acid. Blockade of T-type calcium channels with TTA-P2 (1 μm) or voltage-gated Na+ channels with TTX inhibited BDNF-induced [Ca2+]i increases. BDNF also induced a dose-dependent enhancement of action potential firing in CHox cells. These data demonstrate that during chronic hypoxia, enhancement of BDNF-TrkB signalling increases voltage-dependent Ca2+ influx and catecholamine secretion in chromaffin cells, and that T-type Ca2+ channels play a key role in the signalling pathway. Key points We investigated the role of the neurotrophin BDNF signalling via the TrkB receptor in rat adrenomedullary chromaffin cells (AMCs) exposed to normoxia (Nox; 21% O2) and chronic hypoxia (CHox; 2% O2) in vitro for ∼48 h. TrkB receptor expression was

  13. High revivability of vitrified-warmed bovine mature oocytes after recovery culture with α-tocopherol.

    PubMed

    Yashiro, Ikuko; Tagiri, Miho; Ogawa, Hayato; Tashima, Kazuya; Takashima, Seiji; Hara, Hiromasa; Hirabayashi, Masumi; Hochi, Shinichi

    2015-04-01

    The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

  14. Bovine mammary epithelial cells retain stem-like phenotype in long-term cultures.

    PubMed

    Cravero, Diego; Diego, Cravero; Martignani, Eugenio; Eugenio, Martignani; Miretti, Silvia; Silvia, Miretti; Macchi, Elisabetta; Elisabetta, Macchi; Accornero, Paolo; Paolo, Accornero; Baratta, Mario; Mario, Baratta

    2014-10-01

    The detection and characterization of bovine mammary stem cells may give a better understanding of the cyclic characteristic of mammary gland development. In turn, this could potentially offer techniques to manipulate lactation yield and for regenerative medicine. We previously demonstrated that adult stem cells reside in the bovine mammary gland and possess an intrinsic regenerative potential. In vitro maintenance and expansion of this primitive population is a challenging task that could make easier the study of adult mammary stem cells. The aim of this study is to investigate this possibility. Different subpopulations of mammary epithelial cells emerge when they are cultured in two defined culture conditions. Specific cell differentiation markers as cytokeratin 18 (CK18) and cytokeratin 14 (CK14) were expressed with significant differences according to culture conditions. Vimentin, a well-known fibroblast marker was observed to increase significantly (P < 0.5) only after day 20. In both conditions, after prolonged culture (25 days) a subset of cells still retained regenerative capabilities. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice as shown by the expression of cytokeratin 14 (CK14), cytokeratin 18 (CK18), p63 (a mammary basal cell layer marker) and Epithelial Cell Adhesion Molecule (EpCAM). We also were able to observe the presence of milk proteins signal in these regenerated structures, which is a specific marker of functional mammary alveoli. Progenitor content was also analyzed in vitro through Colony-Forming Cell (CFC) assays with no substantial differences among culture conditions and time points. These results demonstrate that long-term culture of a multipotent cell subpopulation with intrinsic regenerative potential is possible.

  15. Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor.

    PubMed

    Aponte, Pedro M; Soda, Takeshi; van de Kant, H J G; de Rooij, Dirk G

    2006-06-01

    Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A(s)) and paired (A(pr)) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A(s) to A(pr) and spermatogonial chains (A(al)) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.

  16. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    PubMed

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations.

  17. Fresh and vitrified bovine preantral follicles have different nutritional requirements during in vitro culture.

    PubMed

    Castro, S V; Carvalho, A A; Silva, C M G; Santos, F W; Campello, C C; Figueiredo, J R; Rodrigues, A P R

    2014-12-01

    The aim of this study was to compare the efficiency of different media for the in vitro culturing of fresh and vitrified bovine ovarian tissues. Fragments of the ovarian cortex were subjected to vitrification and histological and viability analyses or were immediately cultured in vitro using the alfa minimum essential medium, McCoy's 5A medium (McCoy), or medium 199 (M199). Samples of different culture media were collected on days 1 (D1) and 5 (D5) for quantification of reactive oxygen species and for hormonal assays. In non-vitrified (i.e., fresh) ovarian tissue cultures, the percentage of morphologically normal follicles was significantly greater than that recorded for the other media (e.g., M199). In the case of previously vitrified tissues, the McCoy medium was significantly superior to the other media in preserving follicular morphology up until the last culture day (i.e., D5), thus maintaining a similar percentage from D1 to D5. Reactive oxygen species levels were higher in D1 vitrified cultured tissues, but there were no differences in the levels among the three media after 5 days. The hormonal assays showed that in the case of previously vitrified tissues, at D5, progesterone levels increased on culture in the M199 medium and estradiol levels increased on culture in the McCoy medium. In conclusion, our results indicate that the use of M199 would be recommended for fresh tissue cultures and of McCoy for vitrified tissue cultures.

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

    PubMed

    Goodarzi, Parisa; Arjmand, Babak; Emami-Razavi, Seyed Hassan; Soleimani, Masoud; Khodadadi, Abbas; Mohamadi-Jahani, Fereshteh; Aghayan, Hamid Reza

    2014-01-01

    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.

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

    PubMed

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

    2016-08-02

    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.

  20. Long-term viability and differentiation of bovine oviductal monolayers: bidimensional versus three-dimensional culture.

    PubMed

    Gualtieri, R; Mollo, V; Braun, S; Barbato, V; Fiorentino, I; Talevi, R

    2012-10-15

    Different in vitro models have been developed to study the interaction of gametes and embryos with the maternal tract. In cattle, the interaction of the oviduct with gametes and embryos have been classically studied using oviductal explants or monolayers (OMs). Explants are well differentiated but have to be used within 24 h after collection, whereas OMs can be used for a longer time after cell confluence but dedifferentiate during culture, losing cell polarity and ciliation. Herein, OMs were cultured either in M199 plus 10% fetal calf serum or in a semidefined culture medium (Gray's medium), in an immersed condition on collagen-coated coated microporous polyester or polycarbonate inserts under air-liquid interface conditions. The influence of culture conditions on long-term viability and differentiation of OMs was evaluated through scanning electron microscopy, localization of centrin and tubulin at the confocal laser scanning microscope, and assessment of maintenance of viability of sperm bound to OMs. Findings demonstrated that OMs cultured in an immersed condition with Gray's medium retain a better morphology, do not exhibit signs of crisis at least until 3 wks postconfluence, and maintain the viability of bound sperm significantly better than parallel OMs cultured in M199 plus 10% fetal calf serum. OM culture with Gray's medium in air-liquid interface conditions on porous inserts promotes cell polarity, ciliation, and maintenance of bound sperm viability at least until 3 wks postconfluence. In conclusion, oviduct culture in Gray's medium in an immersed or air-liquid condition allows long-term culture and, in the latter case, also ciliation of bovine OMs, and may represent in vitro systems that mimick more closely the biological processes modulated by the oviduct in vivo. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures.

    PubMed

    Gonzales, Veronica K; de Mulder, Eric L W; de Boer, Trix; Hannink, Gerjon; van Tienen, Tony G; van Heerde, Waander L; Buma, Pieter

    2013-11-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three healthy adult donors. Human meniscal fibrochondrocytes (MFCs) were isolated from resected tissue after a partial meniscectomy on a young patient. Passage-4 MFCs were cultured in monolayer for 24 h, and 3 and 7 days. Six different culture media were used containing different amounts of either PRP or PPP and compared to a medium containing 10% FBS. dsDNA was quantified, and gene expression levels of collagen types I and II and aggrecan were measured at different time points with quantitative polymerase chain reaction in the cultured MFCs. After 7 days, the dsDNA quantity was significantly higher in MFCs cultured in 10% and 20% PRP compared to the other PRP and PPP conditions, but equal to 10% FBS. Collagen type I expression was lower in MFCs cultured with medium containing 5% PRP, 10% and 20% PPP compared to FBS. When medium with 10% PRP or 20% PRP was used, expressions were not significantly different from medium containing 10% FBS. Collagen type II expression was absent in all medium conditions. Aggrecan expression did not show differences between the different media used. However, after 7 days a higher aggrecan expression was measured in most culture conditions, except for 5% PRP, which was similar compared to FBS. Statistical significance was found between donors at various time points in DNA quantification and gene expression, but the same donors were not statistically different in all conditions. At 7 days cell cultured with 10% PRP and 20% PRP showed a higher density, with large areas of clusters, compared to other conditions. In an MFC culture medium, FBS can be replaced by 10% PRP or 20% PRP without altering proliferation and gene expression of human MFCs.

  2. Suboptimal culture conditions induce more deviations in gene expression in male than female bovine blastocysts.

    PubMed

    Heras, Sonia; De Coninck, Dieter I M; Van Poucke, Mario; Goossens, Karen; Bogado Pascottini, Osvaldo; Van Nieuwerburgh, Filip; Deforce, Dieter; De Sutter, Petra; Leroy, Jo L M R; Gutierrez-Adan, Alfonso; Peelman, Luc; Van Soom, Ann

    2016-01-22

    Since the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production. We used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism. Serum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male

  3. Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture

    NASA Astrophysics Data System (ADS)

    Suparto, Irma H.; Khalam, Chandra Nur; Praira, Willy; Sajuthi, Dondin

    2014-03-01

    Fetal Bovine Serum (FBS) in animal cell culture media is an important source of nutrients for cell growth. However, the harvest and collection of FBS cause bioethical concerns. Efforts to reduce and preferably replace FBS with synthetic or other natural alternatives are continually being explored. Hemolymph silkworm (Bombyx mori) contains many nutrients needed for the process of metamorphosis. Therefore, there is possibility as an alternative nutritional supplement for cell culture to reduce the use of FBS. The objective of this study was to evaluate the macrocomponent of hemolymph and the possibility as medium supplement for Spodoptera fugiperda (Sf9) cell culture. Proximate analyses showed that hemolymph contains 89.76% of water, 2.52 mg/mL carbohydrate, 2.35% fat and 55.61 mg/mL protein. Further protein analysis, it consists of 15 fractions containing molecular weight of 22 - 152 kDa. The use of hemolymph as FBS substitution in Sf9 cell culture with various concentrations was unable to maintain and support cell growth. Further research still needed by prior adaptation of the tissue culture to minimal nutrition media before introduction of the hemolymph as supplement.

  4. Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent.

    PubMed

    Jimenez, C R; de Azevedo, J L; Silveira, R G; Penitente-Filho, J; Carrascal-Triana, E L; Zolini, A M; Araújo, V R; Torres, Caa

    2016-08-01

    The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ. © 2016 Blackwell Verlag GmbH.

  5. Perifusion culture system for bovine embryos: improvement of embryo development by use of bovine oviduct epithelial cells, an antioxidant and polyvinyl alcohol.

    PubMed

    Lim, J M; Reggio, B C; Godke, R A; Hansel, W

    1997-01-01

    Three experiments were conducted in an attempt to improve a continuous flow-perifusion system capable of maintaining embryo development for long periods of time. Bovine embryos (8-16 cells) obtained from static co-culture with cumulus cells in a serum-free medium were perifused in an ACUSYST-S cell culture incubator. Culture chambers of the incubator consisted of a 0.2-mL unit (Chamber 1) connected to a 1.5-mL unit (Chamber 2), with the outflow from Chamber 1 routed to the inlet to Chamber 2. A bovine embryo culture medium supplemented with 3 mg mL-1 bovine serum albumin (BSA) and 25 mM HEPES was used as a perifusion culture medium (PCM). Embryos were perifused in Chamber 2 for 24, 48 and 72 h and further co-cultured in a static system up to 216 h after insemination. In Experiment 1, conditioning PCM with frozen-thawed bovine oviduct epithelial cells (BOEC) placed in Chamber 1 enhanced (P < 0.05) blastocyst formation of embryos in Chamber 2, after 24, 48 and 72 h of perifusion culture. The proportion of blastocysts was not further increased by placing BOEC in Chamber 2 along with the embryos. In Experiment 2, embryos were perifused with PCM conditioned with BOEC in Chamber 1 for 48 h or 72 h. A higher proportion of perifused embryos developed to the blastocyst stage after addition of 25 U mL-1 or 50 U mL-1 of superoxide dismutase (SOD) to PCM than in its absence. However, blastocyst formation of embryos perifused for 72 h was not increased after addition of 50 U mL-1 SOD compared with its absence. In Experiment 3, the proportions of morulae and blastocysts were not decreased by replacement of 3 mg mL-1 BSA with 1 mg mL-1 polyvinyl alcohol (PVA) in a BOEC-conditioned medium containing 50 U mL-1 SOD after perifusion for 48 h. In conclusion, PCM conditioning with BOEC and addition of an antioxidant to the perifusion medium improved the developmental capacity of perifused embryos. PVA is an adequate replacement for BSA in the perifusion medium.

  6. Increasing cell plating density mimics an early post-LH stage in cultured bovine granulosa cells.

    PubMed

    Baufeld, Anja; Vanselow, Jens

    2013-12-01

    Cultured ovarian granulosa cells are essential models to study molecular mechanisms of gene regulation during folliculogenesis. Here, we characterize primary tissue culture models for bovine granulosa cells by morphological and physiological parameters and by novel molecular luteinization markers, as transcript abundance and DNA methylation levels. The data show that: (1) collagen substrate increased the number of attached, viable cells; (2) the expression of the key transcripts of estrogen synthesis, CYP19A1, could be induced and maintained in granulosa cells from small to medium but not from large follicles, whereas (3) only granulosa cells from large but not from smaller follicles were responsive to LH; (4) serum supplementation unfavorably transformed the cellular phenotype, induced proliferation and PCNA expression, reduced or abolished the transcript abundance of steroidogenic key genes and of gonadotropin receptor genes, CYP11A1, CYP19A1, FSHR and LHCGR but, however, did not increase the abundance of the luteinization-specific marker transcripts PTGS2, PTX3, RGS2 and VNN2; but (5) by increasing the plating density, estradiol production and the abundance of CYP19A1 transcripts, in particular those derived from the main ovarian promoter P2, were decreased concurrently leaving P2-specific DNA methylation levels unchanged, whereas progesterone secretion was stimulated and the expression of both luteinization-specific marker transcripts, RGS2 and VNN2, was significantly induced. From these data, we conclude that increasing the plating density induces a different, partly complementary, physiological and gene expression profile in cultured bovine granulosa cells and drives the cells towards an early post-LH stage of luteinization, even in the absence of luteinizing agents.

  7. Infection of cultured bovine cells with bovine herpesvirus 1 (BHV-1) or Sendai virus induces different beta interferon subtypes

    PubMed Central

    da Silva, Leticia Frizzo

    2011-01-01

    In contrast to mice or humans, cattle contain three beta interferon (IFN-β) genes with distinct transcriptional promoters suggesting IFN-β gene expression is not stimulated the same by different viruses. To test this hypothesis, we compared expression of the three IFN-β subtypes after infection with a RNA virus, Sendai, versus a large DNA virus, bovine herpesvirus 1 (BHV-1). Infection of low passage bovine kidney (BK) or established bovine kidney cells (CRIB) with Sendai virus has consistently led to high levels of IFN-β1 RNA. Conversely, infection of CRIB cells, but not BK cells, with BHV-1 increased IFN-β3 RNA levels and to a lesser extent the other two IFN-β subtypes. Inhibition of de novo protein synthesis with cycloheximide resulted in higher levels of IFN-β1 and IFN-β2 RNA levels after BHV-1 infection. Further studies demonstrated that BHV-1 immediate early and/or early genes were primarily responsible for inhibiting the IFN response in BK cells. The three bovine IFN-β promoters were cloned upstream of a reporter gene construct, and their properties analyzed in transient transfection assays. Only the IFN-β3 promoter was trans-activated by IRF3 (interferon responsive factor 3). IRF7 and double stranded RNA (polyIC) stimulated IFN-β1 and IFN-β3 promoter activity, but not IFN-β2. Relative to the human IFN-β promoter, the IFN-β3 promoter contained fewer nucleotide differences in the positive regulatory domain III (PRD III), PRD IV, and PRD I compared to the IFN-β1 and IFN-β2 promoter. Collectively, these studies provide evidence that virus infection differentially stimulates expression of the three bovine IFN-β genes. PMID:21316405

  8. Defined media optimization for in vitro culture of bovine somatic cell nuclear transfer (SCNT) embryos.

    PubMed

    Wang, Li-Jun; Xiong, Xian-Rong; Zhang, Hui; Li, Yan-Yan; Li, Qian; Wang, Yong-Sheng; Xu, Wen-Bing; Hua, Song; Zhang, Yong

    2012-12-01

    The objective was to establish an efficient defined culture medium for bovine somatic cell nuclear transfer (SCNT) embryos. In this study, modified synthetic oviductal fluid (mSOF) without bovine serum albumin (BSA) was used as the basic culture medium (BCM), whereas the control medium was BCM with BSA. In Experiment 1, adding polyvinyl alcohol (PVA) to BCM supported development of SCNT embryos to blastocyst stage, but blastocyst formation rate and blastocyst cell number were both lower (P < 0.05) compared to the undefined group (6.1 vs. 32.6% and 67.3 ± 3.4 vs. 109.3 ± 4.5, respectively). In Experiment 2, myo-inositol, a combination of insulin, transferrin and selenium (ITS), and epidermal growth factor (EGF) were added separately to PVA-supplemented BCM. The blastocyst formation rate and blastocyst cell number of those three groups were dramatically improved compared with that of PVA-supplemented group in Experiment 1 (18.5, 23.0, 24.1 vs. 6.1% and 82.7 ± 2.0, 84.3 ± 4.2, 95.3 ± 3.8 vs. 67.3 ± 3.4, respectively, P < 0.05), but were still lower compared with that of undefined group (33.7% and 113.8 ± 3.4, P < 0.05). In Experiment 3, when a combination of myo-inositol, ITS and EGF were added to PVA-supplemented BCM, blastocyst formation rate and blastocyst cell number were similar to that of undefined group (30.4 vs. 31.1% and 109.3 ± 4.4 vs. 112.0 ± 3.6, P > 0.05). In Experiment 4, when blastocysts were cryopreserved and subsequently thawed, there were no significant differences between the optimized defined group (Experiment 3) and undefined group in survival rate and 24 and 48 h hatching blastocyst rates. Furthermore, there were no significant differences in expression levels of H19, HSP70 and BAX in blastocysts derived from optimized defined medium and undefined medium, although the relative expression abundance of IGF-2 was significantly decreased in the former. In conclusion, a defined culture medium containing PVA, myo-inositol, ITS, and EGF

  9. Intraadrenal corticotropin in bilateral macronodular adrenal hyperplasia.

    PubMed

    Louiset, Estelle; Duparc, Céline; Young, Jacques; Renouf, Sylvie; Tetsi Nomigni, Milène; Boutelet, Isabelle; Libé, Rossella; Bram, Zakariae; Groussin, Lionel; Caron, Philippe; Tabarin, Antoine; Grunenberger, Fabienne; Christin-Maitre, Sophie; Bertagna, Xavier; Kuhn, Jean-Marc; Anouar, Youssef; Bertherat, Jérôme; Lefebvre, Hervé

    2013-11-28

    Bilateral macronodular adrenal hyperplasia is a rare cause of primary adrenal Cushing's syndrome. In this form of hyperplasia, hypersecretion of cortisol suppresses the release of corticotropin by pituitary corticotrophs, which results in low plasma corticotropin levels. Thus, the disease has been termed corticotropin-independent macronodular adrenal hyperplasia. We examined the abnormal production of corticotropin in these hyperplastic adrenal glands. We obtained specimens of hyperplastic macronodular adrenal tissue from 30 patients with primary adrenal disease. The corticotropin precursor proopiomelanocortin and corticotropin expression were assessed by means of a polymerase-chain-reaction assay and immunohistochemical analysis. The production of corticotropin and cortisol was assessed in 11 specimens with the use of incubated explants and cell cultures coupled with hormone assays. Corticotropin levels were measured in adrenal and peripheral venous blood samples from 2 patients. The expression of proopiomelanocortin messenger RNA (mRNA) was detected in all samples of hyperplastic adrenal tissue. Corticotropin was detected in steroidogenic cells arranged in clusters that were disseminated throughout the adrenal specimens. Adrenal corticotropin levels were higher in adrenal venous blood samples than in peripheral venous samples, a finding that was consistent with local production of the peptide within the hyperplastic adrenals. The release of adrenal corticotropin was stimulated by ligands of aberrant membrane receptors but not by corticotropin-releasing hormone or dexamethasone. A semiquantitative score for corticotropin immunostaining in the samples correlated with basal plasma cortisol levels. Corticotropin-receptor antagonists significantly inhibited in vitro cortisol secretion. Cortisol secretion by the adrenals in patients with macronodular hyperplasia and Cushing's syndrome appears to be regulated by corticotropin, which is produced by a subpopulation of

  10. Adrenal Histoplasmosis in Immunocompetent Patients Presenting as Adrenal Insufficiency.

    PubMed

    Gajendra, Smeeta; Sharma, Rashi; Goel, Shalini; Goel, Ruchika; Lipi, Lipika; Sarin, Hemanti; Guleria, Mridula; Sachdev, Ritesh

    2016-01-01

    Histoplasmosis is an infectious disease caused by the dimorphic fungus Histoplasma capsulatum, endemic in central and eastern states of United States, South America and Africa. India is considered to be non-endemic area for histoplasmosis. Disseminated histoplasmosis may affect almost all systems. Disseminated histoplasmosis with asymptomatic adrenal involvement has been described in immunocompromised patients; whereas isolated adrenal involvement with adrenal insufficiency as the presenting manifestation of the disease is rare. Twelve patients from a non-endemic area with adrenal histoplasmosis, who were immunocompetent and diagnosed as adrenal histoplasmosis by cytology/histopathology between January 2012 to December 2014 were studied. 18F-FDG PET/CT (fluorodeoxyglucose positron emission tomography/computed tomography) was used to assess the extent of involvement. There were a total of 12 immunocompetent males (mean age: 56.9 years). Ten patients had bilateral adrenal involvement and two had a unilateral left adrenal mass. All the patients had histopathologically/cytologically proven adrenal histoplasmosis. Two patients had simultaneous histoplasmosis of other sites, one in the epiglottis and the other in the alveolus. 18F-FDG PET/CT was performed in 10 patients showing high FDG uptake in the adrenals. All these patients received Amphotericin B and/or Itraconazole treatment that led to symptomatic improvement. A diagnosis of invasive fungal infection requires a high index of suspicion, especially in immunocompetent patients who present with nonspecific symptoms, clinical signs, laboratory and radiological features that can resemble adrenal neoplasms. Clinical specimens must be sent for cytopathology/histopathology together with fungal culture for a definite diagnosis and appropriate management.

  11. Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage

    PubMed Central

    Shil, Sharadindu; Joshi, R. S.; Joshi, C. G.; Patel, A. K.; Shah, Ravi K.; Patel, Namrata; Jakhesara, Subhash J.; Kundu, Sumana; Reddy, Bhaskar; Koringa, P. G.; Rank, D. N.

    2017-01-01

    Aim: Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma) frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn’s SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study. Materials and Methods: Whole transcriptome sequencing of horn’s SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified. Results: Up-regulated genes in SCC of horn’s early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity. Conclusion: The experiment revealed similar transcriptomic nature of horn’s SCC tissue and its early passage cells. PMID:28246447

  12. Responses of bovine early embryos to S-adenosyl methionine supplementation in culture.

    PubMed

    Shojaei Saadi, Habib A; Gagné, Dominic; Fournier, Éric; Baldoceda Baldeon, Luis Manuel; Sirard, Marc-André; Robert, Claude

    2016-08-01

    There is a growing concern about the potential adverse effects of high dose folic acid (FA) supplementation before and during pregnancy. FA metabolism generates S-adenosyl methionine (SAM) which is an important cofactor of epigenetic programming. We sought to assess the impact of a large dose of SAM on early embryo development. In vitro cultured bovine embryos were treated with SAM from the eight-cell stage to the blastocyst stage. In addition to the phenotype, the genome-wide epigenetic and transcription profiles were analyzed. Treatment significantly improved embryo hatching and caused a shift in sex ratio in favor of males. SAM caused genome-wide hypermethylation mainly in exonic regions and in CpG islands. Although differentially expressed genes were associated with response to nutrients and developmental processes, no correspondence was found with the differentially methylated regions, suggesting that cellular responses to SAM treatment during early embryo development may not require DNA methylation-driven changes. Since bovine embryos were not indifferent to SAM, effects of large-dose FA supplements on early embryonic development in humans cannot be ruled out.

  13. Evaluation of Tualang honey as a supplement to fetal bovine serum in cell culture.

    PubMed

    Kannan, Thirumulu Ponnuraj; Ali, Abdulaziz Qaid; Abdullah, Siti Fadilah; Ahmad, Azlina

    2009-07-01

    The aim of this study was to evaluate Tualang honey as a supplement to fetal bovine serum in cell cultures using MTT assay, chromosome aberration test and gene expression analyses. The MTT assay showed the highest percentage of cell proliferation (105.3% increment than control) of human osteoblast cell line (CRL 1543) in 0.0195% honey in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. There was enhanced cell proliferation corresponding to the decrease in concentrations of honey as indicated by the mitotic index values when the osteoblast cell line was incubated at 37 degrees C for 48 hours. There were no chromosome aberrations both in the honey treated as well as distilled water treated (negative control) cell lines. In the case of gene expression analyses, fibroblast cell lines (CCL 171) were treated with honey (0.0195%) for 24 and 48 hours separately. Though there was over expression for the bcl-xl gene at both 24 and 48 hours, under expression for bcl-xs gene at 24 hours and over expression at 48 hours and under expression for both c-myc and p53 genes at both 24 and 48 hours, none of them were statistically significant in altering the expression of mRNA.

  14. Characterization of Silver Nanoparticles in Cell Culture Medium Containing Fetal Bovine Serum.

    PubMed

    Hansen, Ulf; Thünemann, Andreas F

    2015-06-23

    Nanoparticles are being increasingly used in consumer products worldwide, and their toxicological effects are currently being intensely debated. In vitro tests play a significant role in nanoparticle risk assessment, but reliable particle characterization in the cell culture medium with added fetal bovine serum (CCM) used in these tests is not available. As a step toward filling this gap, we report on silver ion release by silver nanoparticles and on changes in the particle radii and in their protein corona when incubated in CCM. Particles of a certified reference material, p1, and particles of a commercial silver nanoparticle material, p2, were investigated. The colloidal stability of p1 is provided by the surfactants polyethylene glycol-25 glyceryl trioleate and polyethylene glycol-20 sorbitan monolaurate, whereas p2 is stabilized by polyvinylpyrrolidone. Dialyses of p1 and p2 reveal that their silver ion release rates in CCM are much larger than in water. Particle characterization was performed with asymmetrical flow field-flow fractionation, small-angle X-ray scattering, dynamic light scattering, and electron microscopy. p1 and p2 have similar hydrodynamic radii of 15 and 16 nm, respectively. The silver core radii are 9.2 and 10.2 nm. Gel electrophoresis and subsequent peptide identification reveal that albumin is the main corona component of p1 and p2 after incubation in CCM that consists of Dulbecco's modified Eagle medium with 10% fetal bovine serum added.

  15. Effects of lipid-related factors on adipocyte differentiation of bovine stromal-vascular cells in primary culture.

    PubMed

    Wu, P; Sato, K; Suzuta, F; Hikasa, Y; Kagota, K

    2000-09-01

    The effects of several factors related to lipids on bovine adipocyte differentiation were investigated in primary culture. Adipocyte differentiation was assessed by development of glycerol-3-phosphate dehydrogenase (GPDH) activity and morphological observation. Addition of triglyceride mixture (Intralipid), caprylic acid and very low-, low- and high-density lipoproteins (VLDL, LDL and HDL) stimulated bovine preadipocyte differentiation in serum-free condition. Especially, VLDL strongly increased both cell protein contents and GPDH activity, suggesting that it stimulated both proliferation and differentiation of bovine preadipocytes. Under Intralipid-induced condition, differentiation of preadipocytes from subcutaneous adipose tissues was more evident than those from omental adipose tissues. However, such depot difference was not observed in medium supplemented with indomethacin, which is a peroxisome proliferator-activated receptor (PPAR) gamma agonist. This suggests that the differentiation capacity of bovine preadipocytes was different between depots and such difference is dependent on the ability to utilize lipids as endogenous PPARgamma ligands. Therefore, lipid metabolites have the stimulatory effects on bovine adipocyte differentiation in vitro, and lipoproteins, especially VLDL, may play an important role in development of bovine adipose tissues in vivo.

  16. Development, molecular composition and freeze tolerance of bovine embryos cultured in TCM-199 supplemented with hyaluronan.

    PubMed

    Palasz, A T; Breña, P Beltrán; Martinez, Marcelo F; Perez-Garnelo, S S; Ramirez, M A; Gutiérrez-Adán, A; De la Fuente, J

    2008-02-01

    Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p<0.05). Following freeze/thawing, significantly more (p<0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p<0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p<0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.

  17. [Adrenal mass and adrenal insufficiency].

    PubMed

    Martínez Albaladejo, M; García López, B; Serrano Corredor, S; Alguacil García, G

    1996-12-01

    Primary adrenal insufficiency is a non frequent disease, that is declared in young adults and in the most of the cases is produced from an autoimmune mechanism or a tuberculous disease. The incidence of these forms in the different geographic areas is dependent of degree of irradication of the tuberculosis. We report the case of a patient with latent chronic adrenal insufficiency of tuberculous origin who was affected for an addisonian crisis during an intercurrent infectious disease, which permitted the diagnosis of the addisonian crisis, and Mal of Pott was moreover detected. Evolution with corticosteroid and specific treatment was very favorable.

  18. Insulin-like growth factor-II stimulates steroidogenesis in cultured bovine thecal cells.

    PubMed

    Spicer, L J; Voge, J L; Allen, D T

    2004-11-30

    The objective of the present study was to determine the effects of insulin-like growth factor-II (IGF-II) on luteinizing hormone (LH)-induced progesterone and androstenedione production by bovine thecal cells and compare it to that of insulin and IGF-I. Cells from large (>7.9 mm) bovine follicles were collected and cultured for 2 days in the presence of 10% fetal calf serum. Then cells were cultured for an additional 1 or 2 days in serum-free medium with various doses of recombinant human IGF-II, bovine LH (30 ng/ml), IGF-I, and(or) insulin. Cell numbers were determined at the end of treatments via Coulter counting and used to correct steroid production data. In the presence of LH, 1-day treatment with 3-300 ng/ml of IGF-II had no significant effect on progesterone or androstenedione production, whereas 2-day treatment with 30, 100 and 300 ng/ml of IGF-II increased (P < 0.05) both progesterone and androstenedione production by 2-3-fold. The estimated effective dose of IGF-II stimulating 50% of the maximal steroidogenic response was calculated to be 25 ng/ml. In the absence of LH, 2-day treatment of IGF-I or -II had no effect on thecal androstenedione production but increased (P < 0.05) thecal progesterone production. In the presence of LH, 100 ng/ml of IGF-I increased progesterone and androstenedione production to a greater degree than did 100 ng/ml of IGF-II. Maximal effects of IGF-I and insulin on thecal steroidogenesis were similar and were not additive. Anti-IGF type I receptor antibodies attenuated (P < 0.05) the stimulatory effect of both IGF-I and IGF-II on thecal cell steroidogenesis. Use of radioligand assays demonstrated that specific receptors for (125)I-IGF-II existed in thecal cells with a 25 ng/well of IGF-II causing 50% inhibition of binding. IGF-I cross-reactivity with (125)I-IGF-II receptors averaged 3% whereas cross-reactivity of IGF-II with (125)I-IGF-I receptors averaged 114%. These results indicate that the stimulatory effects of IGF-II on

  19. Effect of quinine on the release of catecholamines from bovine cultured chromaffin cells.

    PubMed Central

    Tang, R.; Novas, M. L.; Glavinovic, M. I.; Trifaró, J. M.

    1990-01-01

    1. The effects of quinine on catecholamine release from cultured bovine chromaffin cells were studied. 2. Quinine (25-400 microM) produced a dose-related inhibition of catecholamine release in response to depolarizing concentrations (12.5-50 mM) of K+. 3. The inhibition of the secretory response to high K+ produced by quinine decreased with the increase in the extracellular concentration of Ca2+. 4. Stimulation of cultured chromaffin cells with 50 mM K+ produced a significant increase in Ca2+ influx. In the presence of 100 microM quinine a 54% inhibition of the K(+)-induced Ca2+ influx was observed. 5. Quinine treatment of chromaffin cell cultures produced a small but significant decrease in membrane resting potential and a less pronounced depolarization in response to 50 mM K+. 6. The results suggest that the inhibition of the K(+)-evoked release of catecholamines produced by quinine is at least partly due to a decrease in Ca2+ influx. Ca2+ influx is lower because quinine reduces the sensitivity of the membrane potential to changes in extracellular K+ but direct effects of quinine on Ca2+ channels cannot be excluded. PMID:2158846

  20. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    PubMed

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  1. In vitro culture of Babesia bovis in a bovine serum-free culture medium supplemented with insulin, transferrin, and selenite.

    PubMed

    Rojas Martínez, C; Rodríguez-Vivas, R I; Figueroa Millán, J V; Acosta Viana, K Y; Gutiérrez Ruiz, E J; Álvarez Martínez, J A

    2016-11-01

    Bovine serum is an important factor for the optimal growth of Babesia bovis in vitro. This protozoan can be cultured in M-199 with Earle's salts medium (M-199) supplemented with 40% bovine serum (BS). In the present study, four media were assessed along with the control medium M-199. The effect on the proliferation of B. bovis in vitro was tested when these media were combined with insulin (Ins), transferrin (Trans) and selenite (Sel) in the absence of bovine serum. Treatment with Advanced DMEM/F12 medium (A-DMEM/F12) achieved the highest percentage of parasitized erythrocytes (PPE), reaching a maximum value of 9.59%. A-DMEM/F12 medium supplemented with a mixture of Ins (2000 mg/L), Trans (1100 mg/L), and Sel (1.34 mg/L) allowed for the adaptation and proliferation of B. bovis without bovine serum, showed a constant increase in PPE, and reached a maximum value of 9.7% during seven cycles of in vitro culture. It was concluded that continuous proliferation of B. bovis in vitro could be achieved using A-DMEM/F12 medium supplemented with Ins-Trans-Sel, without bovine serum. After adaptation for proliferation in serum-free medium, the B. bovis strain of parasites could have future use in the study of this economically important protozoan species that affects cattle.

  2. Culture of bovine embryos in polyester mesh sections: the effect of pore size and oxygen tension on in vitro development.

    PubMed

    Somfai, T; Inaba, Y; Aikawa, Y; Ohtake, M; Kobayashi, S; Akai, T; Hattori, H; Konishi, K; Imai, K

    2010-12-01

    The purpose of this study was to assess the feasibility of polyester mesh culture for the in vitro production of bovine embryos, as polyester mesh is an alternative way for tracking individual embryos throughout culture using time-lapse cinematography (TLC). Bovine embryos were isolated during in vitro culture using sections of three different polyethylene terephthalate (PET) mesh products. In vitro matured and fertilized bovine oocytes were cultured in the 217 × 217, 230 × 230 or 238 × 238-μm openings of PET mesh sections or in simple micro-drops (control) for 7 days under either 20% or 5% O(2) tensions. No difference in embryo developmental rates was found between the culture groups in terms of cleavage, blastocyst formation and blastocyst expansion irrespective of O(2) tension. In contrast, under 20% O(2) tension, blastocysts that developed in PET mesh with 217 × 217-μm opening had significantly higher numbers of total and trophectoderm (TE) cells than control embryos; however, the numbers and proportions of inner cell mass (ICM) cells did not differ. Under 5% O(2) tension, no difference was found among the culture groups in the numbers of total, ICM and TE cells in embryos. All three PET mesh products investigated in this study were proven to be effective to prevent embryo movement. The results demonstrate that bovine embryos can be cultured in PET mesh sections without negative side-effects and suggest that embryo distance determined by the mesh affects embryo quality at atmospheric oxygen tension. Polyethylene terephthalate mesh with 217 × 217-μm openings was found to be the most suitable for further application in TLC.

  3. 3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.

    PubMed

    Brandt, M; Levy, M A

    1989-01-10

    Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.

  4. Replacing serum in culture medium with albumin and insulin, transferrin and selenium is the key to successful bovine embryo development in individual culture.

    PubMed

    Wydooghe, E; Heras, S; Dewulf, J; Piepers, S; Van den Abbeel, E; De Sutter, P; Vandaele, L; Van Soom, A

    2014-06-01

    Individual culture of bovine embryos is usually associated with low blastocyst development. However, during preliminary experiments in our laboratory we observed high blastocyst development after individual embryo culture in a serum-free culture system. We therefore hypothesised that serum has a negative effect on embryos cultured individually whereas embryos in groups can counteract this. First, we determined whether the timing of removal of serum (during maturation or culture) had an influence on individual embryo development. The results clearly showed that removal of serum during embryo culture was the main contributing factor since high blastocyst development was observed after individual culture in synthetic oviductal fluid supplemented with bovine serum albumin (BSA) and insulin, transferrin and selenium (ITS), independent of the maturation medium. Second, we investigated whether an individual factor of the ITS supplement was essential for individual embryo development. We demonstrated that repeatable high blastocyst percentages were due to the synergistic effect of ITS. Finally, we investigated if a group-culture effect can still be observed under serum-free conditions. Group culture generated blastocysts with higher total cell numbers and less apoptosis. These data show that individual culture in serum-free conditions leads to high blastocyst development, but group culture still improves blastocyst quality.

  5. Elevated non-esterified fatty acid concentrations hamper bovine oviductal epithelial cell physiology in three different in vitro culture systems.

    PubMed

    Jordaens, L; Arias-Alvarez, M; Pintelon, I; Thys, S; Valckx, S; Dezhkam, Y; Bols, P E J; Leroy, J L M R

    2015-10-01

    Elevated non-esterified fatty acids (NEFAs) have been recognized as an important link between lipolytic metabolic conditions and impaired fertility in high-yielding dairy cows. However, NEFA effects on the oviductal micro-environment currently remain unknown. We hypothesize that elevated NEFAs may contribute to the complex pathology of subfertility by exerting a negative effect on bovine oviductal epithelial cell (BOEC) physiology. Therefore, the objectives of this study were to elucidate direct NEFA effects on BOEC physiology in three different in vitro cell culture systems. Bovine oviductal epithelial cells (four replicates) were mechanically isolated, pooled, and cultured as conventional monolayers, as explants, and in a polarized cell culture system with Dulbecco's modified Eagle's medium/F12-based culture medium. Bovine oviductal epithelial cells were exposed to an NEFA mixture of oleic, stearic, and palmitic acids for 24 hours at both physiological and pathologic concentrations. A control (0 μM NEFA) and a solvent control (0 μM NEFA + 0.45% ethanol) group were implemented. Bovine oviductal epithelial cells physiology was assessed by means of cell number and viability, a sperm binding assay, transepithelial electric resistance (TER), and a wound-healing assay. Bovine oviductal epithelial cell morphology was assessed by scanning electron microscopy on cell polarity, presence of microvilli and cilia, and monolayer integrity. Bovine oviductal epithelial cell number was negatively affected by increasing NEFAs, however, cell viability was not. Sperm binding affinity significantly decreased with increasing NEFAs and tended (P = 0.051) to be more affected by the direction of NEFA exposure in the polarized cell culture system. The absolute TER increase after NEFA exposure in the control (110 ± 11 Ω.cm(2)) was significantly higher than that in all the other treatments and was also different depending on the exposure side. Bidirectional exposed monolayers were even

  6. Effects of in vitro growth culture duration and prematuration culture on maturational and developmental competences of bovine oocytes derived from early antral follicles.

    PubMed

    Huang, Weiping; Nagano, Masashi; Kang, Sung-Sik; Yanagawa, Yojiro; Takahashi, Yoshiyuki

    2013-10-15

    Bovine ovaries offer a large pool of oocytes that could be used for in vitro production of embryos of genetically valuable animals. The effects of in vitro growth (IVG) culture duration (10, 12, and 14 days) on the viability and growth of bovine oocytes derived from early antral follicles (0.5-1 mm diameter) in this study. In addition, the effect of pre-IVM culture with phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) on nuclear maturation of IVG oocytes was also evaluated. In experiment 1, oocyte viability observed after 10 or 12 days of IVG culture was greater (P < 0.05) than that observed after 14 days of culture. Oocyte diameters and proportions of oocytes at metaphase II stage were greater (P < 0.05) when 12 or 14 days of IVG culture where used when compared with 10 days culture. In addition, the proportion of oocytes at metaphase II stage was greater (P < 0.05) when pre-IVM culture was performed for oocytes derived from 12 and 14 days of IVG culture. When 12 and 14 days of IVG culture followed by pre-IVM culture were compared in experiment 2, cumulus cell membrane integrity was greater (P < 0.05) after 12 days. Blastocyst production rate for oocytes obtained after 12 days of IVG culture (24.5%) was greater (P < 0.05) than for oocytes obtained after 14 days (9.9%). In conclusion, 12 days IVG followed by pre-IVM culture was considered the optimal processing system for bovine oocytes derived from early antral follicles when oocyte viability, diameter, maturation, and development competences were considered.

  7. In vitro maturation system for individual culture of bovine oocytes using micro-volume multi-well plate.

    PubMed

    Nagano, Masashi; Kang, Sung-Sik; Koyama, Keisuke; Huang, Weiping; Yanagawa, Yojiro; Takahashi, Yoshiyuki

    2013-11-01

    As a preliminary study for the development of individual in vitro maturation (IVM) culture of bovine oocytes, a multi-well (MW) plate was used. Maturation, fertilization and development to blastocysts were examined and compared with those of IVM oocytes cultured in 50-microl droplets in groups and in 10-microl droplets individually. The maturation rates were similar in all experimental groups. Normal fertilization rates in MW and 50-microl droplets were similar, but lower in 10-microl droplets (p < 0.01). The blastocyst rate in 10-microl droplets tended to be lower than those in MW (p = 0.15) and 50-microl droplets (p = 0.19). These results indicate that an IVM system using MW supports the acquisition of developmental competence by bovine oocytes the same as conventional group IVM culture.

  8. Isolation, Characterization, and Differentiation of Progenitor Cells from Human Adult Adrenal Medulla

    PubMed Central

    Santana, Magda M.; Chung, Kuei-Fang; Vukicevic, Vladimir; Rosmaninho-Salgado, Joana; Kanczkowski, Waldemar; Cortez, Vera; Hackmann, Karl; Bastos, Carlos A.; Mota, Alfredo; Schrock, Evelin; Bornstein, Stefan R.; Cavadas, Cláudia

    2012-01-01

    Chromaffin cells, sympathetic neurons of the dorsal ganglia, and the intermediate small intensely fluorescent cells derive from a common neural crest progenitor cell. Contrary to the closely related sympathetic nervous system, within the adult adrenal medulla a subpopulation of undifferentiated progenitor cells persists, and recently, we established a method to isolate and differentiate these progenitor cells from adult bovine adrenals. However, no studies have elucidated the existence of adrenal progenitor cells within the human adrenal medulla. Here we describe the isolation, characterization, and differentiation of chromaffin progenitor cells obtained from adult human adrenals. Human chromaffin progenitor cells were cultured in low-attachment conditions for 10–12 days as free-floating spheres in the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor. These primary human chromosphere cultures were characterized by the expression of several progenitor markers, including nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox9, Sox10, Phox2b, and Ascl1 on the molecular level and of Sox9 on the immunohistochemical level. In opposition, phenylethanolamine N-methyltransferase (PNMT), a marker for differentiated chromaffin cells, significantly decreased after 12 days in culture. Moreover, when plated on poly-l-lysine/laminin-coated slides in the presence of FGF-2, human chromaffin progenitor cells were able to differentiate into two distinct neuron-like cell types, tyrosine hydroxylase (TH)+/β-3-tubulin+ cells and TH−/β-3-tubulin+ cells, and into chromaffin cells (TH+/PNMT+). This study demonstrates the presence of progenitor cells in the human adrenal medulla and reveals their potential use in regenerative medicine, especially in the treatment of neuroendocrine and neurodegenerative diseases. PMID:23197690

  9. Isolation, characterization, and differentiation of progenitor cells from human adult adrenal medulla.

    PubMed

    Santana, Magda M; Chung, Kuei-Fang; Vukicevic, Vladimir; Rosmaninho-Salgado, Joana; Kanczkowski, Waldemar; Cortez, Vera; Hackmann, Klaus; Bastos, Carlos A; Mota, Alfredo; Schrock, Evelin; Bornstein, Stefan R; Cavadas, Cláudia; Ehrhart-Bornstein, Monika

    2012-11-01

    Chromaffin cells, sympathetic neurons of the dorsal ganglia, and the intermediate small intensely fluorescent cells derive from a common neural crest progenitor cell. Contrary to the closely related sympathetic nervous system, within the adult adrenal medulla a subpopulation of undifferentiated progenitor cells persists, and recently, we established a method to isolate and differentiate these progenitor cells from adult bovine adrenals. However, no studies have elucidated the existence of adrenal progenitor cells within the human adrenal medulla. Here we describe the isolation, characterization, and differentiation of chromaffin progenitor cells obtained from adult human adrenals. Human chromaffin progenitor cells were cultured in low-attachment conditions for 10-12 days as free-floating spheres in the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor. These primary human chromosphere cultures were characterized by the expression of several progenitor markers, including nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox9, Sox10, Phox2b, and Ascl1 on the molecular level and of Sox9 on the immunohistochemical level. In opposition, phenylethanolamine N-methyltransferase (PNMT), a marker for differentiated chromaffin cells, significantly decreased after 12 days in culture. Moreover, when plated on poly-l-lysine/laminin-coated slides in the presence of FGF-2, human chromaffin progenitor cells were able to differentiate into two distinct neuron-like cell types, tyrosine hydroxylase (TH)(+)/β-3-tubulin(+) cells and TH(-)/β-3-tubulin(+) cells, and into chromaffin cells (TH(+)/PNMT(+)). This study demonstrates the presence of progenitor cells in the human adrenal medulla and reveals their potential use in regenerative medicine, especially in the treatment of neuroendocrine and neurodegenerative diseases.

  10. Interaction between embryos and culture conditions during in vitro development of bovine early embryos.

    PubMed

    Nagao, Yoshikazu; Iijima, Rumi; Saeki, Kazuhiro

    2008-05-01

    Various factors such as embryo density and substances in the medium can influence embryo development in vitro. These factors and the embryos probably interact with each other, however the interactions are not fully understood. To investigate the interactions, we examined the effects of the number of embryos, drop size, oxygen concentration and glucose and inorganic phosphate in the medium during protein-free culture of bovine IVM/IVF embryos. In Experiment 1, different numbers of embryos were cultured in a 50 microl drop of medium. The frequencies of blastocyst development in the groups with 25, 50 and 100 embryos per drop were higher than in the other groups. One, five and 25 embryos were cultured in different drop sizes (Experiment 2), a 50 microl drop of medium at different O2 concentrations (Experiment 3) and a 50 microl drop of medium excluding glucose and/or inorganic phosphate (Experiment 4). In Experiment 2, the size of the medium drops did not improve blastocyst development. In Experiment 3, the highest frequency of blastocyst development for one, five and 25 embryos per drop was obtained at 1, 2.5 and 5% O2, respectively. In Experiment 4, blastocyst development for one and five embryos per drop were improved in the medium excluded inorganic phosphate. These results indicate that there is a cooperative interaction among embryos during culture and that this interaction may be mediated by reduction of toxic factors in the medium. At low embryo density, reduced oxygen concentration or the exclusion of inorganic phosphate enhanced blastocyst development.

  11. Influence of "Solcoseryl" during culture on the sex-dependent repair of bovine demi-embryos.

    PubMed

    Tominaga, K; Yoneda, K; Utsumi, K

    1996-03-01

    The purpose of this experiment was to determine the effect of culture conditions on the development of split embryos after bisection and on the sex ratio of resultant bovine demi-embryos. Embryos that had developed into blastocysts on days 6 1/2 to 7 or on days 7 1/2 to 8 from oocytes matured and fertilized in vitro were bisected in BMOC-3 medium supplemented with 33% calf serum. The medium also contained 0%, 0.1% or 1.0% Solcoseryl, a deproteinized hemodialysate product from calf blood. The demi-embryos were first cultured for 4 hours in the same medium in which they had been bisected and then co-cultured with cumulus cells in TCM199 supplemented with 1% calf serum for an additional 20 hr. The rate of production of good to excellent quality demi-embryos obtained from days 6 1/2 to 7 blastocysts was higher than from those on days 7 1/2 to 8. The rate was also significantly improved when blastocysts were bisected in medium containing 0.1% or 1.0% Solcoseryl, compared to the medium without Solcoseryl. Male embryos seemed to recover more rapidly than female embryos, as assessed by morphological quality at 4 hr, although the quality of female embryos had improved by 24 hr. The percentage of males after culture was higher in the medium without Solcoseryl than in its presence. Thus, addition of Solcoseryl at either 0.1% or 1.0% to BMOC-3 medium seemed to improve the production efficiency of good quality demi-embryos, but did not influence the sex ratio. It appears as if female demi-embryos required more time than male embryos to be repaired after bisection.

  12. Expression of XIST sense and antisense in bovine fetal organs and cell cultures.

    PubMed

    Farazmand, Ali; Basrur, Parvathi K; Stranzinger, Gerald; Graphodatskaya, Daria; Reyes, Ed R; King, W Allan

    2004-01-01

    Untranslated RNAs transcribed from sense and antisense strands of a gene referred to as X-inactive specific transcript (XIST) play crucial roles in the genetic inactivation and condensation of one of the two X chromosomes in the somatic cells of female mammals. X inactivation is also thought to occur in mammalian male germ cells mainly based on the formation of a condensed structure referred to as a sex body or XY-body, during spermatogenesis. Molecular identity of the sex body, the roles of sense and antisense XIST RNAs in its formation, and the relevance of the sex body to spermatogenesis are not known. Here we report the results of our strand-specific RT-PCR approach to identify the amplicon detected in fetal bovine testes previously referred to as XIST and to test for sense/antisense expression in male and female organs and cell cultures of different sex chromosome constitution. Our results showed that the transcript detected consistently in male gonads and variably in somatic organs represents XIST antisense RNA and that XIST sense and antisense RNAs are co-expressed in female somatic tissues and cultured cells including cells of sex chromosome aneuploids (XXY and XXX). Our results, which differ from those of other investigators in this area, are discussed in the light of the recently reported differences in the expression pattern of murine Xist/Tsix loci and their structural and functional differences in different mammalian species.

  13. Enhanced production of cellobiose dehydrogenase in cultures of Phanerochaete chrysosporium supplemented with bovine calf serum.

    PubMed

    Habu, N; Igarashi, K; Samejima, M; Pettersson, B; Eriksson, K E

    1997-10-01

    The enzyme cellobiose dehydrogenase (CDH), produced by many wood-degrading fungi has, in recent years, attracted considerable interest for its possible role in both cellulose and lignin degradation. To characterize the enzyme better and to identify its role in the degradation of wood and wood components, it is desirable to produce it in higher amounts. We report here that the addition of bovine calf serum to cellulose-grown cultures of Phanerochaete chrysosporium enhances the production of certain enzymes, CDH in particular. The highest CDH production was obtained with 45 ml of serum/litre of medium added on day 3 or 4. The resultant CDH yield was approx. 700-800 units/litre, which was 3.5-4 times higher than that in cultures without serum. Serum addition also enhanced the production of beta-glucosidase. However, the impact on CDH production was the most dramatic. The enhanced enzyme production cannot be explained by increased rates of spore germination, simple nutrient effects or cofactor effects. Fractionation of serum by Cohn's fractionation technique showed that the albumin (BSA) fraction had almost the same effect as whole serum. However, purified BSA had less effect than crude BSA (fraction V of Cohn's fractions), suggesting that an additional factor, probably a protease inhibitor in serum, also contributed to the effect of serum.

  14. L-ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos.

    PubMed

    Zullo, G; Albero, G; Neglia, G; De Canditiis, C; Bifulco, G; Campanile, G; Gasparrini, B

    2016-03-01

    The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of

  15. Inclusion of bovine lipoproteins and the vitamin E analogue, Trolox, during in vitro culture of bovine embryos changes both embryo and fetal development.

    PubMed

    Rooke, J A; Watt, R G; Ashworth, C J; McEvoy, T G

    2012-01-01

    This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100μM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (P<0.001) whereas inclusion of Trolox had no effect (P>0.05). Whereas lipoproteins reduced zygote development to blastocysts (P=0.03), Trolox facilitated increased development (P<0.001) and counteracted the reduction observed with lipoproteins (interaction, P=0.009). Lipoproteins also compromised (P<0.001) but presence of Trolox (P>0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, P<0.001). Liver weight (g kg(-1) fetal weight) was greater (P=0.03) in treatments containing Trolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P=0.002); superior embryo grades were also associated with increased numbers of placentomes (P=0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70.

  16. Bovine oviductal epithelial cells: long term culture characterization and impact of insulin on cell morphology.

    PubMed

    Palma-Vera, S; Einspanier, R; Schoen, J

    2014-09-01

    In vitro models that resemble cell function in vivo are needed to understand oviduct physiology. This study aimed to assess cell functions and insulin effects on bovine oviductal epithelial cells (BOECs) cultured in an air-liquid interface. BOECs (n=6) were grown in conditioned Ham's F12, DMEM or Ham's F12/DMEM with 10% fetal calf serum (FCS) for 3 weeks. After selecting the most suitable medium (Ham's F12), increasing insulin concentrations (1 ng/mL, 20 ng/mL and 5 μg/mL) were applied, and cell morphology and trans-epithelial electrical resistance (TEER; n=4) were evaluated after 3 and 6 weeks. Keratin immunohistochemistry and mRNA expression of oviductal glycoprotein 1 (OVGP1) and progesterone receptor (PGR) were conducted (n=4) to assess cell differentiation. BOECs grown without insulin supplementation or with 1 ng/mL of insulin displayed polarization and secretory activity. However, cells exhibited only 50% of the height of their in vivo counterparts. Cultures supplemented with 20 ng/mL insulin showed the highest quality, but the 5 μg/mL concentration induced massive growth. TEER correlated negatively with insulin concentration (r=-0.459; p=0.009). OVGP1 and PGR transcripts were still detectable after 3 and 6 weeks. Cellular localization of keratins closely resembled that of BOECs in vivo. Cultures showed heterogeneous expression of PGR and OVGP1 in response to estradiol (10 pg/mL). In summary, BOECs grown for long term in an air-liquid interface expressed markers of cell differentiation. Additionally, insulin supplementation (20 ng/mL) improved the cell morphology in vitro.

  17. [Efficiency of bacteriological culture and the immunofluorescent assay to detect Campylobacter fetus in bovine genital fluids].

    PubMed

    Marcellino, Romanela B; Morsella, Claudia G; Cano, Dora; Paolicchi, Fernando A

    2015-01-01

    Bovine genital campylobacteriosis is a reproductive disease that affects cattle production. It is caused by Campylobacter fetus subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). The aim of this study was to identify the presence of C. fetus in genital fluids by bacteriological culture and direct immunofluorescence (DIF) and to compare the results. Two groups of 6 heifers and 5 bulls, one infected with Cff (Cff group) and the other with Cfv (Cfv group) were formed. Two heifers and 2 bulls, all of them uninfected, made up the control group. Samples of cervicovaginal mucus and preputial fluid were processed by culture and DIF. In the Cff group, 100% of the heifers and 80% of the bulls were infected, while in the Cfv group, 50% of the heifers and 60% of the bulls were infected. The degree of agreement (Kappa values) from benchmarking diagnostic techniques were 0.57 for heifers in the Cff group and 0.52 for heifers in the Cfv group, whereas the values for bulls were 0.17 and 0.27, respectively. Heifers yielded more positive results in the DIF assay than in the culture, exhibiting 5.6% increase in the Cff group and 7.4% in the Cfv group. The lowest percentage of positive results for DIF in bulls, 40% less for the Cff group and 5.2% for the Cfv group, could be due to improper sampling. Kappa values showed moderate agreement for the heifers and low for the bulls. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  18. Sterile filtered paraffin oil supports in vitro developmental competence in bovine embryos comparable to co-culture.

    PubMed

    Tae, Jin Cheol; Kim, Eun Young; Lee, Won Don; Park, Se Pill; Lim, Jin Ho

    2006-03-01

    To investigate whether sterile filtered light paraffin oil (SPO) overlaying is superior to washed light mineral oil (WMO) in supporting the in vitro developmental competence of bovine follicular oocytes. In addition, the effects of the two types of oil overlaying were compared with oil overlaying plus co-culture (CC) on bovine embryo development in vitro. Bovine follicular oocytes retrieved from abattoir-derived ovary were in vitro matured, fertilized and cultured in 50 microL drops overlayed with WMO or SPO and were subsequently evaluated for development rates. In second experiment, day 2 embryos grown under WMO overlaying were further cultured for 6 days in the presence (WMO+CC and SPO+CC) or absence of adult ear skin fibroblast-based co-culture system overlaid with WMO or SPO. Blastocysts from each group were evaluated for total nuclei number or were further cultured for 48 h to evaluate post-hatching development. SPO overlaying resulted in significant higher (p < 0.05) development rate to morula (44.8% versus 30.6%) and blastocyst (32.8% versus 21.7%) than WMO. Also, treatment of the day 2 embryo cultures with SPO overlaying or oil plus CC (WMO+CC or SPO+CC groups) reached significantly higher development rates from the morula stage compared to embryo cultures treated with the WMO overlaying (p < 0.05). However, the development rates of the SPO treatment group (morula: 72.7%; blastocyst: 53.1%) were slightly high compared to development of the culture treated with WMO+CC (69.6 and 50.4%, respectively). This similar developmental competence pattern was also observed in cell number and embryo hatching rate. SPO overlaying is superior to WMO and WMO+CC in supporting in vitro development of bovine embryos. The development rates are further enhanced when embryos are cultured in co-culture system overlaid with SPO. Thus, these data suggest that overlaying oil can significantly influence the pre-implantation embryo development in vitro.

  19. Reevaluation of Cl-/HCO3- exchange in cultured bovine corneal endothelial cells.

    PubMed

    Bonanno, J A; Yi, G; Kang, X J; Srinivas, S P

    1998-12-01

    To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport. BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2). Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response. Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused

  20. Uterine tubal cells remain uninfected after culture with in vitro-produced embryos exposed to bovine viral diarrhea virus.

    PubMed

    Givens, M D; Galik, P K; Riddell, K P; Stringfellow, D A

    1999-10-01

    Bovine viral diarrhea virus (BVDV) has been isolated from washed and sonicated, in vitro-produced embryos, but the infectivity of BVDV associated with intact, developing, embryos has not been demonstrated. The objective of this study was to determine if a dose of BVDV infective for co-culture cells was associated with individual, developing embryos, following artificial exposure to the virus and washing. In 5 replicates, zona pellucida-intact, in vitro-produced embryos were assigned to a negative control embryo group, or were incubated in 10(5)-10(6) cell culture infective doses (50%, CCID50) per milliliter of a type I, noncytopathic (strain SD-1) BVDV for 2 h. Unexposed negative control embryos and exposed positive control embryos were washed, sonicated and assayed for BVDV using virus isolation with immunoperoxidase monolayer assay. Immediately or following cryopreservation, remaining virally-exposed, washed embryos were co-cultured individually with BVDV-negative cultures of bovine uterine tubal cells in a medium free of BVDV-neutralizing activity. After two days in culture, uterine tubal cells and embryos (including the zona pellucida) were separated and washed. The culture medium, uterine tubal cells and embryos were then assayed for BVDV. Bovine viral diarrhea virus was not isolated from any negative control embryo group, but was isolated from all positive control embryo groups. Although all uterine tubal cell populations were confirmed to be susceptible to BVDV, virus was never isolated from uterine tubal cells or embryos from post-exposure culture. In conclusion, although BVDV remains associated with washed in vitro-produced embryos, the virus associated with unsonicated embryos was not infective for uterine tubal cells in vitro.

  1. Effects of Fetal Bovine Serum deprivation in cell cultures on the production of Anticarsia gemmatalis Multinucleopolyhedrovirus

    PubMed Central

    2010-01-01

    Background Anticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum. Results Culture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G0/G1 stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells. Conclusions UFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both in vitro bioinsecticide production and applications such as recombinant protein expression or gene delivery. PMID:20843354

  2. Loading-Induced Heat-Shock Response in Bovine Intervertebral Disc Organ Culture

    PubMed Central

    Chooi, Wai Hon; Chan, Samantha Chun Wai; Gantenbein, Benjamin; Chan, Barbara Pui

    2016-01-01

    Mechanical loading has been shown to affect cell viability and matrix maintenance in the intervertebral disc (IVD) but there is no investigation on how cells survive mechanical stress and whether the IVD cells perceive mechanical loading as stress and respond by expression of heat shock proteins. This study investigates the stress response in the IVD in response to compressive loading. Bovine caudal disc organ culture was used to study the effect of physiological range static loading and dynamic loading. Cell activity, gene expression and immunofluorescence staining were used to analyze the cell response. Cell activity and cytoskeleton of the cells did not change significantly after loading. In gene expression analysis, significant up-regulation of heat shock protein-70 (HSP70) was observed in nucleus pulposus after two hours of loading. However, the expression of the matrix remodeling genes did not change significantly after loading. Similarly, expressions of stress response and matrix remodeling genes changed with application and removal of the dynamic loading. The results suggest that stress response was induced by physiological range loading without significantly changing cell activity and upregulating matrix remodeling. This study provides direct evidence on loading induced stress response in IVD cells and contributes to our understanding in the mechanoregulation of intervertebral disc cells. PMID:27580124

  3. Loading-Induced Heat-Shock Response in Bovine Intervertebral Disc Organ Culture.

    PubMed

    Chooi, Wai Hon; Chan, Samantha Chun Wai; Gantenbein, Benjamin; Chan, Barbara Pui

    2016-01-01

    Mechanical loading has been shown to affect cell viability and matrix maintenance in the intervertebral disc (IVD) but there is no investigation on how cells survive mechanical stress and whether the IVD cells perceive mechanical loading as stress and respond by expression of heat shock proteins. This study investigates the stress response in the IVD in response to compressive loading. Bovine caudal disc organ culture was used to study the effect of physiological range static loading and dynamic loading. Cell activity, gene expression and immunofluorescence staining were used to analyze the cell response. Cell activity and cytoskeleton of the cells did not change significantly after loading. In gene expression analysis, significant up-regulation of heat shock protein-70 (HSP70) was observed in nucleus pulposus after two hours of loading. However, the expression of the matrix remodeling genes did not change significantly after loading. Similarly, expressions of stress response and matrix remodeling genes changed with application and removal of the dynamic loading. The results suggest that stress response was induced by physiological range loading without significantly changing cell activity and upregulating matrix remodeling. This study provides direct evidence on loading induced stress response in IVD cells and contributes to our understanding in the mechanoregulation of intervertebral disc cells.

  4. Development and pattern of mRNA relative abundance of bovine embryos cultured in the isolated mouse oviduct in organ culture.

    PubMed

    Rizos, D; Pintado, B; de la Fuente, J; Lonergan, P; Gutiérrez-Adán, A

    2007-06-01

    The aim of this study was to examine the development of bovine zygotes in isolated mouse oviducts (IMO) and the quality of the blastocysts produced. In vitro produced bovine zygotes were transferred into the ampullae of the IMO and cultured in SOF or KSOM. Control embryos were cultured in droplets of the same media. Following 6 days of culture, blastocysts were processed for nuclei counts or mRNA abundance. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 17.7 +/- 3.2% vs. 18.8 +/- 2.7%; KSOM: 20.7 +/- 2.6% vs. 22.2 +/- 2.8%). Culture in the IMO in KSOM resulted in an increased number of inner cell mass (ICM) nuclei; however, total nuclei number or incidence of apoptosis was unaffected. Culture in the IMO in SOF resulted in an increase (P < 0.05) in abundance of transcripts in blastocysts for Oct-4 and SOX, and reduced abundance of Glut-1, Na/K, Cx43, and survivin compared to blastocysts derived from culture in SOF alone. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and reduced expression of Na/K and SOX compared to KSOM alone. Transcripts for G6PDH, IFN-tau, and E-Cad were unaffected. These data confirm that the IMO is capable of supporting development of bovine embryos. Depending on the basal medium used, the pattern of transcript abundance in embryos derived from the IMO is similar to that of in vivo derived embryos.

  5. Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer Yellow in cultured bovine oviductal cells.

    PubMed

    Tiemann, U; Pöhland, R

    1999-01-01

    The present study investigated the effects of dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and gamma-hexachlorocyclohexane (gammaHCH, lindane) on gap junctional intercellular communication (GJIC) in cultured bovine oviductal cells. GJIC was evaluated by microinjecting fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. After incubation for 1 h at 37 degrees C, a dose-dependent inhibition of GJIC was observed over a concentration range of 16 to 128 microM DDT, MXC, or gammaHCH compared with nonexposed controls. A significant inhibition began at 32 microM DDT, MXC, or gammaHCH. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 microM of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 microM DDT, 16 microM MXC, and 32 microM gammaHCH. The 128 microM concentration of the pesticides was toxic. At pesticide concentration of 64 microM, the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 microM DDT from the culture medium, intercellular communication was reestablished within 3 h. Measurement of cytosolic free Ca2+ concentration [Ca2+]i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC, or gammaHCH was not associated with a detectable increase in [Ca2+]i. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 microM DDT-induced inhibition of intercellular communication in adherent oviduct cells. It is suggested that organochlorine pesticides can influence cells responsible for reproduction.

  6. Metastatic tumors to the adrenal glands in domestic animals.

    PubMed

    Labelle, P; De Cock, H E V

    2005-01-01

    Although metastases to the adrenals are common in humans, they have not been thoroughly studied in animals. The purpose of this retrospective study was to document the types of malignant tumors that metastasize to canine, feline, equine, and bovine adrenals, and the rate at which they do so. The average rate of adrenal involvement in metastatic cancer was 112/534 (21.0%) in dogs, 12/81 (14.8%) in cats, 18/67 (26.9%) in horses, and 5/16 (31.3%) in cattle. In dogs, 26 different tumor types metastasized to the adrenals. Pulmonary, mammary, prostatic, gastric, and pancreatic carcinomas, and melanoma had the highest rates of metastasis to the adrenal glands in dogs. Hemangiosarcoma and melanoma had high rates of adrenal involvement in horses. In cats and cattle, relevant data were only available for lymphoma. Adrenal metastases usually occurred in the late stages of the disease. One dog had developed Addison's disease (hypoadrenocorticism) secondary to lymphoma. Metastatic lesions represented 126/472 (26.7%) of canine, 12/20 (60.0%) of feline, 21/80 (26.3%) of equine, and 5/9 (55.5%) of bovine adrenal neoplasms. This study shows that adrenal glands should be thoroughly examined during both clinical work-up and postmortems when disseminated neoplasia is suspected.

  7. Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes.

    PubMed

    Opiela, Jolanta; Latasiewicz, Ewa; Smorag, Zdzisław

    2012-12-01

    With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 mL volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 microL dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn't affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn't have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes' meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells.

  8. [Studies on the biosynthesis and secretion of parathyroid hormone in monolayer cultures of bovine parathyroid cells (I) (author's transl)].

    PubMed

    Okano, K; Nakai, R; Goto, H; Yoshikawa, M

    1976-11-20

    We have developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3 yields to 0.5 percent collagenase at 37 degrees C for 60 min. Dispersed cells were cultured at 37 degrees C in MEM-Hanks' BSS containing 10 percent fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1 percent BSA-MEM-Hanks-HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10(-7) yields to 10(-5)M in the former and 10(-5) yields to 10(-3)M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3 yields to 4.4 mM.

  9. Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

    PubMed

    Taylor, Drew W; Ahmed, Nazish; Hayes, Anthony J; Ferguson, Peter; Gross, Allan E; Caterson, Bruce; Kandel, Rita A

    2012-08-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

  10. Adrenal metabolism of mitotane and related compounds

    SciTech Connect

    Djanegara, T.K.S.

    1989-01-01

    Mitotane (o,p{prime}-DDD; 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane) has been used in the treatment of Cushing's syndrome due to adrenal hyperfunction and it the drug of choice for adrenocortical carcinoma. The object of this investigation is to study the biotransformation of o,p{prime}-DDD and p,p{prime}-DDD in dogs and bovine adrenal cortex to explain its selective toxicity and mechanism of action. The in vitro biotransformation of {sup 14}C-labeled o,p{prime}-DDD and p,p{prime}-DDD by dog and bovine adrenal cortex as studied. Of the cortex subcellular fractions, the cytosol fraction was found to be the most active in metabolizing the substrates, followed by the mitochondrial fraction. This metabolism including that in cytosolic fractions, did not take place with boiled enzyme preparations and required an NADPH generating system. This study has been directed towards establishing the metabolic activation mechanism which may account for the adrenocorticolytic effect of mitotane in contrast to detoxication by the liver. HPLC and TLC metabolic profiles have been generated from incubations of bovine and dog adrenal cortex homogenates and their subfractions for {sup 14}C-labeled p,p{prime}-DDD, o,p{prime}-DDD and its monochloroethylene derivative, o,p{prime}-DDMU.

  11. Morphological changes in cultured bovine lymphoid cell lines associated with bovine viral diarrhea virus (BVDV) single and dual infections with bovine leukemia virus (BLV)

    USDA-ARS?s Scientific Manuscript database

    Currently, American Type Culture Collection (ATCC) makes available two cell lines derived from the same lymphoblast-like suspension cell that have been confirmed by next-generation sequencing and RT-PCR to have either a single contaminate of BVDV2a (CRL-8037) or dual contaminates of both BVDV and BL...

  12. Adrenal gland disorders.

    PubMed

    Berry, Matthew E

    2009-01-01

    Medical imaging of the adrenal glands is an important aspect of the diagnosis of any adrenal gland disorder. This article discusses the normal anatomy and functions of the adrenal glands, as well as specific adrenal gland disorders and how they are diagnosed and treated. Radiologic technologists need to understand the causes, signs, symptoms, diagnosis and management of disorders that prevent the adrenal glands from functioning properly.

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

    PubMed Central

    Ülker, Hayriye Esra; Ülker, Mustafa; Gümüş, Hasan Önder; Yalçın, Muhammet; Şengün, Abdulkadir

    2013-01-01

    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 (P < 0.05). 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 (P > 0.05). 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. PMID:23984419

  14. Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture.

    PubMed

    Smith, Sadie L; Everts, Robin E; Sung, Li-Ying; Du, Fuliang; Page, Raymond L; Henderson, Boyd; Rodriguez-Zas, Sandra L; Nedambale, Tshimangadzo L; Renard, Jean-Paul; Lewin, Harris A; Yang, Xiangzhong; Tian, X Cindy

    2009-01-01

    In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.

  15. Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

    PubMed

    Santana, Priscila Di Paula Bessa; Silva, Thiago Velasco Guimarães; da Costa, Nathália Nogueira; da Silva, Bruno Barauna; Carter, Timothy Frederick; Cordeiro, Marcela da Silva; da Silva, Bruno José Martins; Santos, Simone do Socorro Damasceno; Herculano, Anderson Manoel; Adona, Paulo Roberto; Ohashi, Otávio Mitio; Miranda, Moysés dos Santos

    2014-10-01

    Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.

  16. Effect of resveratrol supplementation during culture on the quality and cryotolerance of bovine in vitro produced embryos.

    PubMed

    Salzano, A; Albero, G; Zullo, G; Neglia, G; Abdel-Wahab, A; Bifulco, G; Zicarelli, L; Gasparrini, B

    2014-12-30

    The aim of the study was to evaluate whether resveratrol supplementation of bovine culture medium improves in vitro blastocyst development, embryo cryotolerance and cell numbers. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, zygotes were cultured in SOF medium, supplemented with 0 (control, n=439), 0.25μM (n=422), 0.5μM (n=447) and 1μM resveratrol (n=416). On Day 7 (IVF=Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5M sucrose. Development rate, i.e. the percentage of embryos resuming development to reach a more advanced stage, and hatching rate were evaluated after 24 and 48h culture. Blastocysts cultured with (0.5μM) and without resveratrol underwent differential staining to count inner cell mass (ICM) and trophectoderm (TE) cells. Resveratrol during culture did not increase blastocyst yields (57.1, 57.7, 59.2 and 46.6%, respectively in 0, 0.25, 0.5 and 1μM resveratrol). However, 0.5μM resveratrol improved embryo cryotolerance compared to the control, as indicated by higher development rates (67.3% vs 50.3%, respectively; P<0.01) and hatching rates (58.9% vs 30.9%, respectively; P<0.01) recorded after 48h post-warming culture. Blastocysts produced in the control and in 0.5μM resveratrol groups had similar numbers of ICM (34.1 and 36.4, respectively), TE (88.1 and 85.3, respectively) and total (122.2 and 121.7, respectively) cells. In conclusion, low levels of resveratrol during in vitro culture improve the quality of IVP bovine embryos, as indicated by their increased resistance to cryopreservation.

  17. Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro.

    PubMed

    Reyes-Moreno, Carlos; Boilard, Mathieu; Sullivan, Robert; Sirard, Marc-André

    2002-12-01

    Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture

  18. Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells.

    PubMed

    Kuang, Kunyan; Li, Yansui; Yiming, Maimaiti; Sánchez, José M; Iserovich, Pavel; Cragoe, E J; Diecke, Friedrich P J; Fischbarg, Jorge

    2004-07-01

    The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH

  19. Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells.

    PubMed

    Gupta, Sandeep K; Haigh, Brendan J; Seyfert, Hans-Martin; Griffin, Frank J; Wheeler, Thomas T

    2017-03-01

    Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.

  20. Establishment of a 3D cell culture model of primary bovine mammary epithelial cells extracted from fresh milk.

    PubMed

    Hillreiner, Maria; Müller, Nadine I; Koch, Heiner M; Schmautz, Christiane; Küster, Bernhard; Pfaffl, Michael W; Kliem, Heike

    2017-06-22

    For the investigation of molecular processes underlying diseases of the bovine mammary gland, primary bovine mammary epithelial cells (pbMEC) are used. They are known to contribute to the innate immune system of the bovine mammary gland. The functionality of pbMEC depends on the maintenance of in vivo characteristics. So far, the optimization of pbMEC culture conditions was intended in a variety of experiments. For this purpose, most of the studies used stable cell lines or primary cells obtained from udder biopsies of slaughtered animals. By contrast, within our study, pbMEC of healthy and first lactating Brown Swiss cows were non-invasively isolated from fresh milk. The non-invasively isolated pbMEC were cultivated on the extracellular matrix-like scaffold Matrigel®. Further, they were challenged with different compositions of proliferation media, containing lactogenic hormones and/or the essential amino acid L-lysine. Changes in expression levels of genes coding for milk proteins and for components of the janus kinase/signal transducers and activators of transcription (JAK-STAT) and mTOR pathways were analyzed by RT-qPCR. The secreted proteins were analyzed by LC-MS/MS measurements. We showed for the first time the establishment of a physiologically functional 3D cell culture model of pbMEC isolated from fresh milk. This represents a primary cell culture model system, based on non-invasive cell collection, that can be used to unravel physiological processes in an unbiased manner.

  1. Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

    PubMed

    Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

    2014-10-01

    The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

  2. Activation of spinal MrgC-Gi-NR2B-nNOS signaling pathway by Mas oncogene-related gene C receptor agonist bovine adrenal medulla 8-22 attenuates bone cancer pain in mice.

    PubMed

    Sun, Yu'e; Zhang, Juan; Lei, Yishan; Lu, Cui'e; Hou, Bailing; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    In the present study, we investigate the effects of Mas oncogene-related gene (Mrg) C receptors (MrgC) on the expression and activation of spinal Gi protein, N-methyl-D-aspartate receptor subunit 2B (NR2B), and neuronal nitric oxide synthase (nNOS) in mouse model of bone cancer pain. The number of spontaneous foot lift (NSF) and paw withdrawal mechanical threshold (PWMT) were measured after inoculation of tumor cells and intrathecal injection of MrgC agonist bovine adrenal medulla 8-22 (BAM8-22) or MrgC antagonist anti-MrgC for 14 days after operation. Expression of spinal MrgC, Gi protein, NR2B and nNOS and their phosphorylated forms after inoculation was examined by immunohistochemistry and Western blotting. Double labeling was used to identify the co-localization of NR2B or nNOS with MrgC in spinal cord dorsal horn (SCDH) neurons. The effects of intrathecal injection of BAM8-22 or anti-MrgC on nociceptive behaviors and the corresponding expression of spinal MrgC, Gi protein, NR2B and nNOS were also investigated. The expression of spinal MrgC, Gi protein, NR2B, and nNOS was higher in tumor-bearing mice in comparison to sham mice or normal mice. Intrathecal injection of MrgC agonist BAM8-22 significantly alleviated bone cancer pain, up-regulated MrgC and Gi protein expression, and down-regulated the expression of spinal p-NR2B, t-nNOS and p-nNOS in SCDH on day 14 after operation, whereas administration of anti-MrgC produced the opposite effect. Meanwhile, MrgC-like immunoreactivity (IR) co-localizes with NR2B-IR or nNOS-IR in SCDH neurons. The present study demonstrates that MrgC-activated spinal Gi-NR2B-nNOS signaling pathway plays important roles in the development of bone cancer pain. These findings may provide a novel strategy for the treatment of bone cancer pain.

  3. Activation of spinal MrgC-Gi-NR2B-nNOS signaling pathway by Mas oncogene-related gene C receptor agonist bovine adrenal medulla 8-22 attenuates bone cancer pain in mice

    PubMed Central

    Sun, Yu’e; Zhang, Juan; Lei, Yishan; Lu, Cui’e; Hou, Bailing; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    Objectives: In the present study, we investigate the effects of Mas oncogene-related gene (Mrg) C receptors (MrgC) on the expression and activation of spinal Gi protein, N-methyl-D-aspartate receptor subunit 2B (NR2B), and neuronal nitric oxide synthase (nNOS) in mouse model of bone cancer pain. Methods: The number of spontaneous foot lift (NSF) and paw withdrawal mechanical threshold (PWMT) were measured after inoculation of tumor cells and intrathecal injection of MrgC agonist bovine adrenal medulla 8-22 (BAM8-22) or MrgC antagonist anti-MrgC for 14 days after operation. Expression of spinal MrgC, Gi protein, NR2B and nNOS and their phosphorylated forms after inoculation was examined by immunohistochemistry and Western blotting. Double labeling was used to identify the co-localization of NR2B or nNOS with MrgC in spinal cord dorsal horn (SCDH) neurons. The effects of intrathecal injection of BAM8-22 or anti-MrgC on nociceptive behaviors and the corresponding expression of spinal MrgC, Gi protein, NR2B and nNOS were also investigated. Results: The expression of spinal MrgC, Gi protein, NR2B, and nNOS was higher in tumor-bearing mice in comparison to sham mice or normal mice. Intrathecal injection of MrgC agonist BAM8-22 significantly alleviated bone cancer pain, up-regulated MrgC and Gi protein expression, and down-regulated the expression of spinal p-NR2B, t-nNOS and p-nNOS in SCDH on day 14 after operation, whereas administration of anti-MrgC produced the opposite effect. Meanwhile, MrgC-like immunoreactivity (IR) co-localizes with NR2B-IR or nNOS-IR in SCDH neurons. Conclusions: The present study demonstrates that MrgC-activated spinal Gi-NR2B-nNOS signaling pathway plays important roles in the development of bone cancer pain. These findings may provide a novel strategy for the treatment of bone cancer pain. PMID:27158400

  4. Enhancement in colonization of bovine spermatogonial stem cells following addition of knock-out serum replacement to culture medium

    PubMed Central

    Youssefi, Reza; Tajik, Parviz; Movahedin, Mansoureh; Akbarinejad, Vahid

    2016-01-01

    Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Knock-out serum replacement (KSR) has been reported to enhance the proliferation of murine SSCs and human embryonic stem cells. The aim of the present study was to investigate the effect of KSR versus fetal bovine serum (FBS) and their interaction on colonization of bovine SSCs in vitro. When FBS (10%) was replaced with KSR (10%), a significant increase in the colonization of SSCs and the expression of Thy1, as marker for enrichment of SSCs, was observed. It was revealed that the lesser proliferative effect of FBS as well as the greater proliferative impact of KSR on SSCs colonization were not irreversible as cells having been cultured with FBS (10%) for three days with low colonization showed high rate of colonization in response to KSR (10%) and cells having been cultured with KSR (10%) with high colonization experienced low rate of colonization in response to FBS (10%). Further, it was shown that FBS did not contain factors inhibiting SSCs colonization and it simply lacked factors essential for SSCs proliferation because the combination of FBS (5%) and KSR (5%) resulted in even greater rate of colonization than did KSR (10%). In conclusion, the present study showed that addition of KSR to culture medium would significantly increase SSCs proliferation. PMID:28144417

  5. Enhancement in colonization of bovine spermatogonial stem cells following addition of knock-out serum replacement to culture medium.

    PubMed

    Youssefi, Reza; Tajik, Parviz; Movahedin, Mansoureh; Akbarinejad, Vahid

    2016-01-01

    Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Knock-out serum replacement (KSR) has been reported to enhance the proliferation of murine SSCs and human embryonic stem cells. The aim of the present study was to investigate the effect of KSR versus fetal bovine serum (FBS) and their interaction on colonization of bovine SSCs in vitro. When FBS (10%) was replaced with KSR (10%), a significant increase in the colonization of SSCs and the expression of Thy1, as marker for enrichment of SSCs, was observed. It was revealed that the lesser proliferative effect of FBS as well as the greater proliferative impact of KSR on SSCs colonization were not irreversible as cells having been cultured with FBS (10%) for three days with low colonization showed high rate of colonization in response to KSR (10%) and cells having been cultured with KSR (10%) with high colonization experienced low rate of colonization in response to FBS (10%). Further, it was shown that FBS did not contain factors inhibiting SSCs colonization and it simply lacked factors essential for SSCs proliferation because the combination of FBS (5%) and KSR (5%) resulted in even greater rate of colonization than did KSR (10%). In conclusion, the present study showed that addition of KSR to culture medium would significantly increase SSCs proliferation.

  6. Bovine Oviduct Epithelial Cells Dedifferentiate Partly in Culture, While Maintaining their Ability to Improve Early Embryo Development Rate and Quality.

    PubMed

    Schmaltz-Panneau, B; Locatelli, Y; Uzbekova, S; Perreau, C; Mermillod, P

    2015-10-01

    There are convincing arguments to suggest that the success of early reproductive events is reliant on a satisfactory dialogue between gametes-embryo and the oviduct epithelium. The aim of this study was to develop and characterize an in vitro model to study these interactions. Cattle zygotes produced in vitro were cultured in either SOF or TCM-199 in the presence or absence of bovine oviduct cell monolayers (BOEC), under 20% or 5% O2 . The embryonic development rate and its quality (cell numbers, cryosurvival) were evaluated, as were the BOEC contents in 11 candidate transcripts (real-time PCR) at different time points. A BOEC co-culture did indeed increase the rate of development in both media under 5% O2 (41 vs 27% and 28 vs 10% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). The effect of BOEC on the developmental rate was more pronounced under 20% O2 (35 vs 6% and 27 vs 4% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). BOEC significantly increased the embryonic cell count in TCM-199 (122.5 ± 11.1 vs 70.3 ± 9.6; p < 0.05) and embryonic cryosurvival in both media. The expression levels of SOD, FGF2 and TGF-β1 in BOEC remained steady during culture, although mRNA levels of OGP, C3, PGR and ESR2 were clearly reduced, suggesting a dedifferentiation of BOEC during culture. However, SSP1 and GPX4 transcripts were slightly increased during culture, this rise becoming significant by the end of the culture period. In conclusion, our co-culture system with bovine oviduct epithelial cells used for the development of bovine zygotes produced in vitro enhanced blastocyst formation and above all the quality of the resulting embryos, which was associated with specific transcriptomic changes.

  7. Neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands.

    PubMed

    Bozhok, G A; Sidorenko, O S; Plaksina, E M; Gurina, T M; Sukach, A N; Kholodnyy, V S; Ustichenko, V D; Bilyavskaya, S B; Bondarenko, T P; Legach, E I

    2016-10-01

    Stem/progenitor cells are thought to have the potential in the treatment of severe neurodegenerative diseases. Recently, sympathoadrenal progenitors expressing specific markers of neural crest derivatives and capable to differentiate into neurons were discovered in adult bovine and human adrenal glands, but there was no reported data on cryopreservation of sympathoadrenal progenitors. The aim of the present study was to examine the neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands. Considering impact of various initial state of frozen biomaterial on cell recovery, we carried out a comparative estimation of cryopreservation outcome both for adrenal tissue fragments and isolated primary cells. The estimation consisted of determining cell yield, viability, ability to adhere, proliferate and differentiate in vitro. Cells isolated from the fresh adrenal glands were cultured until confluence. A formation of sympathoadrenal progenitors-embedded spherical cell colonies, whose cells are differentiated then into βIII-tubulin-positive cells with neuron-like morphology, was observed on the monolayer. The colonies were well preserved after cryopreservation of cell culture with a cooling rate of 1 °C/min in the cryoprotectant media containing 5-15% of dimethylsulfoxide. Adrenal tissue fragments were cryopreserved in the presence of 10% dimethylsulfoxide at the cooling rates of 0.3; 1: 5; 40 and > 100 °C/min. Sympathoadrenal progenitors were recovered after cryopreservation with 0.3 °C/min cooling rate but not higher. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Adrenal Steroidogenesis and Congenital Adrenal Hyperplasia

    PubMed Central

    Turcu, Adina F.; Auchus, Richard J.

    2015-01-01

    Synopsis Adrenal steroidogenesis is a dynamic process, reliant on de novo synthesis from cholesterol, under the stimulation of ACTH and other regulators. The syntheses of mineralocorticoids, glucocorticoids and adrenal androgens occur in separate adrenal cortical zones, each expressing specific enzymes. Congenital adrenal hyperplasia (CAH) encompasses a group of autosomal recessive enzymatic defects in cortisol biosynthesis. 21-hydroxylase (21OHD) deficiency accounts for over 90% of CAH cases and when milder or nonclassic forms are included, 21OHD is one of the most common genetic diseases. This review discusses in detail the epidemiology, genetics, diagnostic, clinical aspects and management of 21OHD. PMID:26038201

  9. Bacterial community profiling of milk samples as a means to understand culture-negative bovine clinical mastitis.

    PubMed

    Kuehn, Joanna S; Gorden, Patrick J; Munro, Daniel; Rong, Ruichen; Dong, Qunfeng; Plummer, Paul J; Wang, Chong; Phillips, Gregory J

    2013-01-01

    Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1-V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease.

  10. Sodium-calcium exchange in cultured bovine pulmonary artery endothelial cells.

    PubMed Central

    Sage, S O; van Breemen, C; Cannell, M B

    1991-01-01

    1. Intracellular free calcium ([Ca2+]i) was measured in cultured bovine pulmonary artery endothelial cell monolayers loaded with the fluorescent calcium indicator Fura-2. 2. Resting [Ca2+]i was 112 +/- 10 nM. Application of ouabain (20 microM) was without effect on [Ca2+]i for periods of up to 1 h. Monensin (10 microM) resting [Ca2+]i to 145 +/- 32 nM over approximately 2 min. In the presence of ouabain (20 microM), 10 microM-monensin increased [Ca2+]i to 146 +/- 15 nM. 3. Removal of extracellular sodium was without effect in resting cells or cells exposed to ouabain alone. However, in the presence of monensin, replacement of extracellular Na+ with Li+ resulted in a prompt increase in [Ca2+]i to a peak of 280 +/- 37 nM, which then returned towards resting levels. When Na+ was removed in the presence of both ouabain and monensin, [Ca2+]i reached a peak of 585 +/- 53 nM. 4. When extracellular Na+ was replaced with K+, to achieve simultaneous Na+ removal and depolarization, [Ca2+]i reached a peak of 568 +/- 63 nM, compared with a peak of 462 +/- 38 nM when Li+ was used as a Na+ substitute in paired experiments. The transient increase in [Ca2+]i evoked by sodium removal peaked earlier when K+ was used as the sodium substitute, showing that depolarization increased the rate of calcium influx into the cell when sodium was removed from the bathing medium. 5. Removal of extracellular K+ had no effect on the low-Na(+)-evoked increase in [Ca2+]i. 6. Returning extracellular Na+ during the increase in [Ca2+]i resulting from Na+ removal increased the rate of return of [Ca2+]i towards basal levels. In the absence of Na+, [Ca2+]i took 41 +/- 5 s to decline from 400 to 200 nM, and this was reduced to 26 +/- 6 s (n = 4, S.E.M.) when Na+ was returned to the bathing solution. 7. These results indicate endothelial cells possess a voltage-dependent Na(+) -Ca2+ exchange mechanism in the surface membrane. However, this mechanism does not appear to be of primary importance in the

  11. Bradykinin-stimulated calcium influx in cultured bovine aortic endothelial cells

    SciTech Connect

    Schilling, W.P.; Ritchie, A.K.; Navarro, L.T.; Eskin, S.G. Univ. of Texas Medical Branch, Galveston )

    1988-08-01

    Bradykinin (BK)-stimulated release of endothelium-derived relaxing factor has been linked to a rise in cytosolic Ca{sup 2+} concentration and a change of K{sup +} permeability of the endothelial cell. In the present study, measurement of BK-induced changes in fura-2 fluorescence and {sup 86}Rb{sup +} efflux were used to monitor changes in cytosolic Ca{sup 2+} and K{sup +} permeability in cultured bovine aortic endothelial cells. In the presence of normal extracellular Ca{sup 2+}, BK induced a fourfold increase in cytosolic Ca{sup 2+}, which peaked at 20 s and declined within 1 min to a value that was 50% of the peak level. Subsequently, cytosolic Ca{sup 2+} decreased and approached basal levels within 8 min. In the absence of Ca{sup 2+}, BK produced a 1.5- to 2-fold increase in cytosolic Ca{sup 2+} that peaked within 20 s and declined to basal levels within 2 min. Addition of Ca{sup 2+} to the Ca-free reaction buffer 3-5 min after addition of BK resulted in a two-to three-fold increase in cytosolic Ca{sup 2+} that declined slowly back to basal levels. Thus Ca{sup 2+} influx can occur in response to BK at a time when there is minimal elevation of cytosolic Ca{sup 2+} above the resting level. Under all conditions tested, {sup 86}Rb{sup +} efflux paralleled changes in the cytosolic Ca{sup 2+}, suggesting that efflux occurred through Ca{sup 2+}-activated K{sup +} channels. Isosmotic substitution of Na{sup +} with N-methyl-D-glucamine did not affect the BK-stimulated changes in cytosolic Ca{sup 2+} or {sup 86}Rb{sup +} efflux, suggesting that Na{sup +}-Ca{sup 2+} exchange plays little role in the BK response. These results suggest that BK stimulates Ca{sup 2+} influx via a BK receptor-operated channel or a channel activated by some internal messenger other than Ca{sup 2+}.

  12. Assay of bovine interferons in cultures of the porcine cell line IB-RS-2.

    PubMed Central

    Ahl, R; Rump, A

    1976-01-01

    An assay for bovine interferons has been developed using the porcine cell line IB-RS-2 and a bovine enterovirus, CBV-D, as challenge virus. The method is based on estimation of cytopathic effect measured by uptake of neutral red. Teh assay is simple, sensitive, and reproducible. A comparative test of different viruses in IB-RS-2 cells and secondary calf kidney cells revealed that the sensitivity of a virus to interferon can vary up to 1,000-fold in the two cell systems. Vesicular stomatitis virus was found to be rather insensitive to interferon in IB-RS-2 cells. PMID:184049

  13. Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos.

    PubMed

    Nicacio, Alessandra Corallo; Simões, Renata; de Paula-Lopes, Fabiola Freitas; de Barros, Flavia Regina Oliveira; Peres, Maria Angelica; Assumpção, Mayra Elena Ortiz D'Avila; Visintin, Jose Antonio

    2012-05-01

    The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

  14. Effects of proteins on the permeability of monolayers of cultured bovine arterial endothelium.

    PubMed Central

    Turner, M R

    1992-01-01

    1. Monolayers of arterial endothelium on porous membranes were exposed to a pressure of 15 cmH2O at 37 degrees C, or of 30 cmH2O at 0 degree C. At constant pressure, the rate of liquid flow per unit area (Jv/A) through each monolayer decreased with time, in the way previously described for cultured endothelium. This phenomenon has been called sealing. After Jv/A stabilized, the pressure was reduced and the hydraulic permeability (Lp) of the endothelium was calculated from the relationship between Jv/A and pressure. Endothelium was seen to be damaged after some experiments at 37 degrees C, but appeared undamaged after experiments at 0 degree C. 2. Bovine serum albumin (BSA) did not influence the Lp of cultured endothelium. At 37 degrees C, the mean (+/- S.E.M.) endothelial Lp was 47.2 +/- 7.3 x 10(-7) cm s-1 cmH2O-1 (n = 10) in the presence of BSA (5 g (100 ml)-1). This is not significantly different from the mean (+/- S.E.M.) Lp of 53.4 +/- 9.0 x 10(-7) cm s-1 cmH2O-1 (n = 9) in the absence of added protein (P greater than 0.10). At 0 degree C also, there was no significant difference between mean Lps in the presence of BSA (0.1 g (100 ml)-1) and in the absence of added protein. 3. Solutions of BSA (5 g (100 ml)-1 or of the neutral polymer Ficoll 70 (4 g (100 ml)-1) did not exert any effective osmotic pressure across endothelium at 37 or 0 degrees C, respectively. 4. BSA (0.1 g (100 ml)-1) did not enable solutions of Ficoll 70 (4 g (100 ml)-1) to exert an effective osmotic pressure across endothelium at 0 degree C. 5. The mean Lp of endothelium at 0 degree C was significantly lower in the presence of cationized ferritin (CF; 0.1 g (100 ml)-1) than in the absence of added protein (P less than 0.001). Native ferritin (NF; 0.1 g (100 ml)-1) had no effect on Lp. 6. In the presence of CF (0.1 g (100 ml)-1), solutions of Ficoll 70 (4 g (100 ml)-1) exerted a mean effective osmotic pressure of 27.7 cmH2O (n = 5) across endothelium at 0 degree C. The mean effective osmotic

  15. Adrenal Gland Disorders

    MedlinePlus

    ... adrenal gland disorders include Genetic mutations Tumors including pheochromocytomas Infections A problem in another gland, such as the pituitary, which helps to regulate the adrenal gland Certain medicines Treatment depends on which problem you have. Surgery or ...

  16. Adrenal Gland Tumors: Statistics

    MedlinePlus

    ... Gland Tumor: Statistics Request Permissions Adrenal Gland Tumor: Statistics Approved by the Cancer.Net Editorial Board , 03/ ... primary adrenal gland tumor is very uncommon. Exact statistics are not available for this type of tumor ...

  17. Adrenal Gland Cancer

    MedlinePlus

    ... either benign or malignant. Benign tumors aren't cancer. Malignant ones are. Most adrenal gland tumors are ... and may not require treatment. Malignant adrenal gland cancers are uncommon. Types of tumors include Adrenocortical carcinoma - ...

  18. Acute adrenal crisis

    MedlinePlus

    ... adrenal gland is damaged due to, for example, Addison disease or other adrenal gland disease, and surgery The ... Call your health care provider if you have Addison disease and are unable to take your glucocorticoid medicine ...

  19. Usage of a selective media (EMJH-STAFF) in primary culturing of pathogenic leptospires from bovine clinical samples.

    PubMed

    Loureiro, A P; Martins, G; Pinto, P; Narduche, L; Teixeira, R C; Lilenbaum, W

    2015-12-01

    Isolation of local strains is mandatory for the success of control programs. However, clinical samples are typically contaminated by other bacteria, which impair leptospires growth. The purpose of this study was to evaluate the use of a previously reported EMJH-STAFF media in the recovery of pathogenic leptospires from bovine clinical samples, namely urine (n = 123) and vaginal fluid-VF (n = 102). EMJH-STAFF presented less contamination than EMJH (<0·005), which was more evident in VF culture tubes. Nine pure leptospires cultures were obtained, six from urine (4·9%) and three from VF (2·9%). From those, seven grew on EMJH-STAFF, one on EMJH and one in both media. All the isolates were confirmed as pathogenic leptospires by lipL32-PCR, and sequencing of partial rrs showed them to belong to Leptospira noguchii, Leptospira santarosai and Leptospira interrogans species. EMJH-STAFF media was an important tool in the recovery of leptospires from bovine clinical samples. The slow growth of leptospires and overgrowth of co-existing micro-organisms from environmental and microbiota are the major difficult to recovery Leptospira from animal clinical samples. Implementing an efficient control programme is essential to determine circulating leptospires in the region and their reservoirs. This study evaluated the relationship of a selective media (EMJH-STAFF) on the recovery of pathogenic leptospires (Leptospira noguchii, Leptospira santarosai and Leptospira interrogans), from bovine clinical samples (urine and vaginal fluid). EMJH-STAFF seems to be an important tool in obtaining local strains for epidemiological and control purposes. © 2015 The Society for Applied Microbiology.

  20. Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

    PubMed Central

    1981-01-01

    Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures. PMID:6454694

  1. [Comparison of human cord blood mesenchymal stem cell culture between using human umbilical cord plasma and using fetal bovine serum].

    PubMed

    Ding, Yan; Lu, Zhiyong; Yuan, Yahong; Wang, Xiaoli; Li, Dongsheng; Zeng, Yi

    2013-12-01

    To investigate whether human umbilical cord plasma (HUP) can be used to culture human cord blood mesenchymal stem cells (HUCMSCs), we collected 20 surplus HUP. After being treated with salting out and diasysis, the HUP were used to culture HUCMSCs as 10% volume, and compared with fetal bovine serum (FBS). Morphological characteristics, growth curve and reproductive activity of HUCMSCs cells were observed. The concentration of bFGF and noggin secreted by HUCMSCs cultured with HUP and FBS medium were detected by ELISA. It was found that compared to FBS, the morphology, reproductive activity and characteristic of HUCMSCs cell cultured with HUP were not distinctively different from FBS. The concentration of bFGF in HUP group was significantly higher than that of FBS group, and the concentration of noggin was also different in the two groups. So we concluded that HUP could be used to culture HUCMSCs for a long-time, and the HUP mediumcoild could be more suitable for the culture of human embryonic stem cell (hESC).

  2. Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro.

    PubMed

    Takahashi, Toshikiyo; Inaba, Yasushi; Somfai, Tamas; Kaneda, Masahiro; Geshi, Masaya; Nagai, Takashi; Manabe, Noboru

    2013-01-01

    High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

  3. In vitro developmental competence of in vitro-matured bovine oocytes fertilized and cultured in completely defined media.

    PubMed

    Keskintepe, L; Brackett, B G

    1996-08-01

    The objective was to establish an in vitro system in which bovine oocytes can be matured, fertilized, and cultured up to the blastocyst stage without support of serum, BSA, or somatic cells. Media consisted of modified tissue culture medium 199 (mTCM 199) with ovine LH (oLH) for maturation (IVM), experimental alterations of modified defined medium (mDM) for sperm selection and insemination (IVF), and citrate+synthetic oviductal fluid+nonessential amino acids (c-SOF+NEA) for zygote/embryo culture (IVC). Effects of heparin, BSA, polyvinyl alcohol (PVA), penicillamine (P), Hepes, and sodium bicarbonate (NaHCO3) were studied. Results included proportions of oocytes that cleaved by 48 h and that reached morulae by 120 h, blastocysts by 168 h, and expanded blastocysts by 216 h postinsemination (pi). Best results were obtained when the IVF medium included 0.5 mg P+1.0 mg PVA per milliliter with no more than 10 mM Hepes, and when 3.0 mg PVA/ml and 10 mM Hepes were present for IVC. Different concentrations of NaHCO3, up to 50 mM from 25 mM, during IVF did not alter results. Embryos produced in defined conditions yielding the best results remained viable after vitrification as evidenced by continued development in vitro for 96 h postthawing. Bovine oocytes matured in defined medium supplemented with LH were fertilized and cultured up to the blastocyst stage in chemically defined conditions that afforded results comparable to those reported earlier after inclusion of serum, BSA, and/or somatic cells.

  4. Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions.

    PubMed

    Vasconcelos, R B; Salles, L P; Oliveira e Silva, I; Gulart, L V M; Souza, D K; Torres, F A G; Bocca, A L; Rosa e Silva, A A M

    2013-08-01

    Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

  5. Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

    PubMed Central

    Vasconcelos, R.B.; Salles, L.P.; Silva, I. Oliveira e; Gulart, L.V.M.; Souza, D.K.; Torres, F.A.G.; Bocca, A.L.; Silva, A.A.M. Rosa e

    2013-01-01

    Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures. PMID:23969977

  6. Short communication: effect of vitamins E and C on cortisol production by bovine adrenocortical cells in vitro.

    PubMed

    Montalvo, C P; Díaz, N H; Galdames, L A; Andrés, M E; Larraín, R E

    2011-07-01

    The aim was to determine if vitamins E and C inhibit the release of cortisol from bovine adrenocortical cells when stimulated with ACTH in vitro. A factorial arrangement of treatments was used to culture bovine adrenocortical cells with different concentrations of vitamins E and C [(+)-α-tocopherol at 0, 2.3, and 16 μM and l-ascorbic acid at 0, 15, and 50 μM]. After 3 and 7 d of vitamin treatments, cell cultures were stimulated with ACTH (1 nM) for 24h and the culture medium extracted to measure cortisol released by the cells using HPLC with UV detection. Vitamin E, vitamin C, or their combination did not affect the amount of cortisol released by the adrenal cultures to the media. Cortisol released by the adrenal cultures ranged from 33.6±6.85 to 49.7±8.01 nmol per 10(7) cells. The modulation effect of vitamins E and C on the stress response does not take place at the cortex of the adrenal gland. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

    PubMed Central

    2011-01-01

    Background Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N. Methods In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison. Results Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. Conclusion Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving. PMID:21244651

  8. Quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation.

    PubMed

    Zhang, Xinsheng; Hasoksuz, Mustafa; Spiro, David; Halpin, Rebecca; Wang, Shiliang; Vlasova, Anastasia; Janies, Daniel; Jones, Leandro R; Ghedin, Elodie; Saif, Linda J

    2007-06-20

    The genetic diversity of 2 pairs (AH65 and AH187) of wild type bovine coronaviruses (BCoV) sequenced directly from nasal (respiratory) and rectal (enteric) swabs of two feedlot calves with respiratory and enteric symptoms [Hasoksuz, M., Sreevatsan, S., Cho, K.O., Hoet, A.E., Saif, L.J., 2002b. Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates. Virus Res. 84 (1-2), 101-109.]. was analyzed. Sequence analysis of the complete genomes revealed differences at 123 and 149 nucleotides (nt) throughout the entire genome between the respiratory and enteric strains for samples AH65 and AH187, respectively, indicating the presence of intra-host BCoV quasispecies. In addition, significant numbers of sequence ambiguities were found in the genomes of some BCoV-R and BCoV-E strains, suggesting intra-isolate quasispecies. The tissue culture (TC) passaged counterparts of AH65 respiratory BCoV (AH65-R-TC) and enteric BCoV (AH65-E-TC) were also sequenced after 14 and 15 passages and 1 plaque purification in human rectal tumor cells (HRT-18), respectively. Compared to the parental wild type strains, tissue culture passage generated 104 nt changes in the AH65-E-TC isolate but only 8 nt changes in the AH65-R-TC isolate. Particularly noteworthy, the majority of nucleotide changes in the AH65-E-TC isolate occurred at the identical positions as the mutations occurring in the AH65-R strain from the same animal. These data suggest that BCoV evolves through quasispecies development, and that enteric BCoV isolates are more prone to genetic changes and may mutate to resemble respiratory BCoV strains after tissue culture passage.

  9. [Pediatric emergency: adrenal insufficiency and adrenal crisis].

    PubMed

    Martínez, Alicia; Pasqualini, Titania; Stivel, Mirta; Heinrich, Juan Jorge

    2010-04-01

    Adrenal insufficiency is defined by impaired secretion of adrenocortical hormones. It is classified upon the etiology in primary and secondary. Rapid recognition and therapy of adrenocortical crisis are critical to survival. Patients often have nonspecific symptoms: anorexia, vomiting, weakness, fatigue and lethargy. They are followed by hypotension, shock, hypoglicemia, hyponatremia and hyperkalemia. All patients with adrenal insufficiency require urgent fluid reposition, correction of hypoglycemia and glucocorticoid replacement, in order to avoid serious consequences of adrenal crisis. After initial crisis treatment, maintenance dose of corticoids should be indicated. Mineralocorticoids replacement, if necessary, should also be initiated.

  10. Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples

    PubMed Central

    Parker, Alysia M.; House, John K.; Hazelton, Mark S.; Bosward, Katrina L.; Sheehy, Paul A.

    2017-01-01

    Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required. PMID:28264012

  11. Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples.

    PubMed

    Parker, Alysia M; House, John K; Hazelton, Mark S; Bosward, Katrina L; Sheehy, Paul A

    2017-01-01

    Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required.

  12. Bacterial Community Profiling of Milk Samples as a Means to Understand Culture-Negative Bovine Clinical Mastitis

    PubMed Central

    Kuehn, Joanna S.; Gorden, Patrick J.; Munro, Daniel; Rong, Ruichen; Dong, Qunfeng; Plummer, Paul J.; Wang, Chong; Phillips, Gregory J.

    2013-01-01

    Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1–V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease. PMID:23634219

  13. Transcriptomal profiling of bovine ovarian granulosa and theca interna cells in primary culture in comparison with their in vivo counterparts

    PubMed Central

    Hatzirodos, Nicholas; Glister, Claire; Hummitzsch, Katja; Irving-Rodgers, Helen F.; Knight, Philip G.; Rodgers, Raymond J.

    2017-01-01

    In vitro culture of ovarian granulosa cells and theca cells has been very important for our understanding of their function and regulation. One of the most eagerly sought attributes of cell culture is the use of chemically-defined conditions. However, even under such in vitro conditions cell behaviour could differ from the in vivo situation because of differences in oxygen tension, nutrients, adhesion matrix and other factors. To examine this further we compared the transcriptomes of both granulosa cells and cells from the theca interna that were cultured in what are arguably the best in vitro conditions for maintaining the ‘follicular’ phenotypes of both tissue types, as displayed by their respective freshly-isolated counterparts. The array data analysed are from recently published data and use the same sizes of bovine follicles (small antral 3–6 mm) and the same Affymetrix arrays. We conducted analysis using Partek, Ingenuity Pathway Analysis and GOEAST. Principal Component Analysis (PCA) and hierarchical clustering clearly separated the in vivo from the in vitro groups for both cells types and transcriptomes were more homogeneous upon culture. In both cell cultures behaviours associated with cell adhesion, migration and interaction with matrix or substrate were more abundant. However, the pathways involved generally differed between the two cell types. With the thecal cultures a gene expression signature of an immune response was more abundant, probably by leukocytes amongst the cells cultured from the theca interna. These results indicate differences between in vivo and in vitro that should be considered when interpreting in vitro data. PMID:28282394

  14. Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition.

    PubMed

    Van Soom, A; Yuan, Y Q; Peelman, L J; de Matos, D G; Dewulf, J; Laevens, H; de Kruif, A

    2002-03-15

    Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.

  15. High density lipoproteins stimulate the production and secretion of endothelin-1 from cultured bovine aortic endothelial cells.

    PubMed

    Hu, R M; Chuang, M Y; Prins, B; Kashyap, M L; Frank, H J; Pedram, A; Levin, E R

    1994-03-01

    The concentration of HDL in the blood inversely correlates with the incidence of cardiovascular disease, probably related to the ability of these lipoproteins to efflux cholesterol from vascular cells. it is also possible that HDL affect the production or action of vasoactive peptides implicated in the development of vascular diseases. Therefore, we determined the effects of human HDL on the production and secretion of endothelin-1 (ET-1) from cultured bovine aortic endothelial cells. HDL produced a highly significant stimulation of endothelin secretion (maximum 240% of control), even at very low levels of lipoproteins (1 microgram/ml). HDL also stimulated the translation of ET-1 by twofold in the bovine aortic endothelial cells. In contrast, HDL had no significant effect on steady state mRNA levels, transcript degradation, or transcription. Stimulation of ET-1 secretion by HDL was dependent on protein kinase C activation. Purified apo A-I, the major apoprotein of HDL, increased ET-1 secretion and translation approximately 85% as potently as HDL. Our results indicate that low concentrations of human HDL strongly stimulate the production of ET-1, a powerful vasoconstrictor and mitogen for the vascular smooth muscle cell. We propose that HDL may participate in the regulation of vasomotor tone through this potentially important effect in the vasculature.

  16. Effect of supplemented sericin on the development, cell number, cryosurvival and number of lipid droplets in cultured bovine embryos.

    PubMed

    Hosoe, Misa; Inaba, Yasushi; Hashiyada, Yutaka; Imai, Kei; Kajitani, Kenji; Hasegawa, Yuichi; Irie, Mamoru; Teramoto, Hidetoshi; Takahashi, Toru; Niimura, Sueo

    2017-02-01

    Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P < 0.05). At 72 h after thawing, the cryosurvival rate of the blastocysts developed using 5% FBS and 0.05% sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P < 0.05). The blastocysts developed using 0.05% sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible.

  17. A Serum Neutralization Test for Infectious Bovine Rhinotracheitis Based on Colour Reaction and Cytopathic effects in Cell Culture

    PubMed Central

    Greig, A. S.

    1969-01-01

    A serum neutralization (SN) test based on a combination of indicator colour change in medium and cytopathic (CP) effect in cells has been devised for the detection of infectious bovine rhinotracheitis antibodies. Serum dilutions of 1:6, 1:18 and 1:54 are made in a medium containing phenol red and are mixed in equal quantities with a suspension of virus containing 100 cell culture infectious doses (CCID50) per volume of mixture. The serum-virus mixtures are held in small glass tubes and are covered with a layer of mineral oil. Following a two hour period of incubation at 37°C a quantity of bovine fetal kidney cells is added to each tube to detect the presence of unneutralized virus. After four to six days incubation the results of the SN test may be read by microscopic examination for CP effect by means of an inverted microscope, or by observing the colour of the phenol red. PMID:4305762

  18. Culture of Primary Bovine Chondrocytes on a Continuously Expanding Surface Inhibits Dedifferentiation

    PubMed Central

    Rosenzweig, Derek H.; Matmati, Mourad; Khayat, Ghazaleh; Chaudhry, Sidharth; Hinz, Boris

    2012-01-01

    Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies. PMID:22738340

  19. Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation.

    PubMed

    Rosenzweig, Derek H; Matmati, Mourad; Khayat, Ghazaleh; Chaudhry, Sidharth; Hinz, Boris; Quinn, Thomas M

    2012-12-01

    Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies.

  20. Bovine chromaffin cells in culture show carboxylesterase activities sensitive to organophosphorus compounds.

    PubMed

    Sogorb, M A; Vilanova, E; Quintanar, J L; Viniegra, S

    1996-09-01

    Carboxylesterase activities are widely distributed in a great variety of tissues; however, the biological function of these enzymes remains unclear. Some organophosphorus compounds induce a neurodegenarative syndrome related to the covalent modification of a carboxylesterase known as neuropathy target esterase. We investigated the expression of neuropathy target esterase and related carboxylesterase in bovine chromaffin cells with the aim of developing a potential in vitro model for studying the cellular function of carboxylesterase enzymes and toxic effects of organophosphorus compounds. Total phenyl valerate esterase exhibited an activity of 1.27 +/- 0.19 mU/10(5) cells (SD, n = 15). From the phenyl valerate esterase paraoxon and mipafox inhibition curves the following activities have been determined: B-activity (resistant to 40 microM paraoxon), 1.05 +/- 0.08 mU/10(5) cells (n = 8); C-activity (resistant to 40 microM paraoxon plus 250 microM mipafox), 0.12 +/- 0.05 mU/10(5) cells (n = 8); and neuropathy target esterase, calculated by the difference between B- and C-activities, 0.93 +/- 0.08 mU/10(5) cells (n = 8). All of these activities increased linearly with the number of cells and time of incubation with the substrate. Most of the phenol product of the reaction was released and detected in the extracellular medium. None of the components of the reaction were shown to affect cell viability when assessed by trypan blue exclusion. The study shows that bovine chromaffin cells possess carboxylesterase activities and respond to inhibition by paraoxon and mipafox, thus facilitating the discrimination of neuropathy target esterase. In conclusion, bovine chromaffin cells are appropriate as an in vitro cell model for studying toxic effects of organophosphorus compounds.

  1. Effect of diet on ability of Vascular Endothelial Growth Factor A (VEGFA) isoforms to alter follicular progression in bovine ovarian cortical cultures

    USDA-ARS?s Scientific Manuscript database

    The objective of this study was to determine the effect of changes in diet on ability of VEGFA isoforms to alter follicle progression in bovine ovarian cortex cultures. Our hypothesis was that diet would affect the magnitude of VEGFA isoform actions on follicular development. Heifers (n = 30) receiv...

  2. The agonistic adrenal: melatonin elicits female aggression via regulation of adrenal androgens.

    PubMed

    Rendon, Nikki M; Rudolph, Lauren M; Sengelaub, Dale R; Demas, Gregory E

    2015-11-22

    Classic findings have demonstrated an important role for sex steroids as regulators of aggression, but this relationship is lacking within some environmental contexts. In mammals and birds, the adrenal androgen dehydroepiandrosterone (DHEA), a non-gonadal precursor of biologically active steroids, has been linked to aggression. Although females, like males, use aggression when competing for limited resources, the mechanisms underlying female aggression remain understudied. Here, we propose a previously undescribed endocrine mechanism regulating female aggression via direct action of the pineal hormone melatonin on adrenal androgens. We examined this in a solitary hamster species, Phodopus sungorus, in which both sexes are highly territorial across the seasons, and display increased aggression concomitant with decreased serum levels of sex steroids in short 'winter-like' days. Short- but not long-day females had increased adrenal DHEA responsiveness co-occurring with morphological changes in the adrenal gland. Further, serum DHEA and total adrenal DHEA content were elevated in short days. Lastly, melatonin increased DHEA and aggression and stimulated DHEA release from cultured adrenals. Collectively, these findings demonstrate that DHEA is a key peripheral regulator of aggression and that melatonin coordinates a 'seasonal switch' from gonadal to adrenal regulation of aggression by direct action on the adrenal glands.

  3. The agonistic adrenal: melatonin elicits female aggression via regulation of adrenal androgens

    PubMed Central

    Rudolph, Lauren M.; Sengelaub, Dale R.; Demas, Gregory E.

    2015-01-01

    Classic findings have demonstrated an important role for sex steroids as regulators of aggression, but this relationship is lacking within some environmental contexts. In mammals and birds, the adrenal androgen dehydroepiandrosterone (DHEA), a non-gonadal precursor of biologically active steroids, has been linked to aggression. Although females, like males, use aggression when competing for limited resources, the mechanisms underlying female aggression remain understudied. Here, we propose a previously undescribed endocrine mechanism regulating female aggression via direct action of the pineal hormone melatonin on adrenal androgens. We examined this in a solitary hamster species, Phodopus sungorus, in which both sexes are highly territorial across the seasons, and display increased aggression concomitant with decreased serum levels of sex steroids in short ‘winter-like' days. Short- but not long-day females had increased adrenal DHEA responsiveness co-occurring with morphological changes in the adrenal gland. Further, serum DHEA and total adrenal DHEA content were elevated in short days. Lastly, melatonin increased DHEA and aggression and stimulated DHEA release from cultured adrenals. Collectively, these findings demonstrate that DHEA is a key peripheral regulator of aggression and that melatonin coordinates a ‘seasonal switch’ from gonadal to adrenal regulation of aggression by direct action on the adrenal glands. PMID:26582025

  4. Bovine ovarian stem cells differentiate into germ cells and oocyte-like structures after culture in vitro.

    PubMed

    de Souza, G B; Costa, Jjn; da Cunha, E V; Passos, Jrs; Ribeiro, R P; Saraiva, Mva; van den Hurk, R; Silva, Jrv

    2017-04-01

    Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM(+) supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte-like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers. © 2016 Blackwell Verlag GmbH.

  5. Low serum concentration in bovine embryo culture enhances early blastocyst rates on Day-6 with quality traits in the expanded blastocyst stage similar to BSA-cultured embryos.

    PubMed

    Murillo, A; Muñoz, M; Martín-González, D; Carrocera, S; Martínez-Nistal, A; Gómez, E

    2017-06-01

    In bovine, single in vitro embryo culture in protein-free medium from Day-6 to Day-7 leads to expanded blastocyst (XB) with improved pregnancy and birth rates after cryopreservation. Under these conditions, early blastocysts (EB) progress to the XB stage at higher rates than morulae (M). However, embryo production with BSA in culture prior to Day-6 leads to low EB rates. We investigated whether a very low FCS concentration (0.1%) in culture from Day-1 to Day-6 would improve EB rates and, subsequently, increase XB rates on Day-7 after single culture in protein-free medium. The quality of embryos produced was evaluated in terms of survival to cryopreservation, apoptosis percentage, lipid accumulation and transfer to recipients. On Day-6, EB rates from embryos cultured with FCS were higher than with BSA (P=0.022). On Day-7, XB rates were higher in embryos from Day-6 EB than from Day-6M, both with and without FCS (P<0.005). After vitrification/warming of Day-7 XB, 100% embryos survived at 24h in all treatments, and total cell number and apoptosis percentage were not affected by the presence of FCS or embryonic stage on Day-6. Cryopreserved and fresh embryos produced with FCS until Day-6, and then deprived of protein and cultured individually, led to pregnancies after ET. In conclusion, minute FCS concentration improves EB rates on Day-6 leading, after one-day single culture without protein, to more XBs. The quality of XB produced with FCS compares well with XB produced with BSA in terms of apoptosis, lipid accumulation and pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  6. Contribution of oocyte source and culture conditions to phenotypic and transcriptomic variation in commercially produced bovine blastocysts.

    PubMed

    Plourde, Dany; Vigneault, Christian; Lemay, Alexandra; Breton, Lévéke; Gagné, Dominic; Laflamme, Isabelle; Blondin, Patrick; Robert, Claude

    2012-07-01

    Bovine embryo production is practiced worldwide for commercial purposes. A major concern of embryo suppliers is the impact of in vitro production systems on embryo quality. In the present study, we compared Buffalo Rat Liver cell coculture with semidefined, medium-based culture, oocytes recovered postmortem with those obtained from live animals, and in vitro with in vivo embryo development. Gene expression levels in expanded blastocysts were measured using microarray and quantitative RT-PCR. The systems were similar in terms of blastocyst yield and rate of development, whereas embryo productivity was greater for immature oocytes collected in vivo. Although immature oocytes collected in vivo had greater developmental competence, they yielded blastocysts that were indistinguishable (in terms of level of gene expression) from embryos derived from immature oocytes recovered postmortem. Culture conditions had a significant impact on gene expression, particularly among genes involved in lipid metabolism. Numerous uncharacterized novel transcript regions were also influenced by in vitro treatments. In conclusion, ovum pick-up combined with in vitro culture in semidefined medium provided a high blastocyst yield, without the deleterious effects associated with coculture.

  7. Isolation of neural crest derived chromaffin progenitors from adult adrenal medulla.

    PubMed

    Chung, Kuei-Fang; Sicard, Flavie; Vukicevic, Vladimir; Hermann, Andreas; Storch, Alexander; Huttner, Wieland B; Bornstein, Stefan R; Ehrhart-Bornstein, Monika

    2009-10-01

    Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Unlike the closely-related sympathetic neurons, a subpopulation of proliferation-competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation-competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine-N-methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo-2'-deoxyuridine-incorporation analysis demonstrated the self-renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinson's disease.

  8. Altered gene expression in human adipose stem cells cultured with fetal bovine serum compared to human supplements.

    PubMed

    Bieback, Karen; Ha, Viet Anh-Thu; Hecker, Andrea; Grassl, Melanie; Kinzebach, Sven; Solz, Hermann; Sticht, Carsten; Klüter, Harald; Bugert, Peter

    2010-11-01

    Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by polymerase chain reaction and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change ≥2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change ≤0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix-associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analyzing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation-associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-specific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo

  9. Congenital Adrenal Hyperplasia

    PubMed Central

    Speiser, Phyllis W.

    2015-01-01

    Congenital adrenal hyperplasia associated with deficiency of steroid 21-hydroxylase is the most common inborn error in adrenal function and the most common cause of adrenal insufficiency in the pediatric age group. As patients now survive into adulthood, adult health-care providers must also be familiar with this condition. Over the past several years, F1000 has published numerous commentaries updating research and practical guidelines for this condition. The purposes of this review are to summarize basic information defining congenital adrenal hyperplasia and to highlight current knowledge and controversies in management. PMID:26339484

  10. cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA.

    PubMed Central

    Russell, D W; Yamamoto, T; Schneider, W J; Slaughter, C J; Brown, M S; Goldstein, J L

    1983-01-01

    The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of approximately equal to 5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of approximately equal to 5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns. Images PMID:6143315

  11. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    USGS Publications Warehouse

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

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  12. Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells.

    PubMed

    Miranda, Moysés S; Nascimento, Hamilton S; Costa, Mayra P R; Costa, Nathália N; Brito, Karynne N L; Lopes, Cinthia T A; Santos, Simone S D; Cordeiro, Marcela S; Ohashi, Otávio M

    2016-10-01

    Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 10(3) and 10(4) cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 10(4) b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). In experiment 1, co-culture with 10(4) b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 10(3) b-ATMSCs/mL (p = 0.051), group 10(4) b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 10(4) b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in

  13. Evaluation of a simple Theileria annulata culture protocol from experimentally infected bovine whole blood.

    PubMed

    Gharbi, M; Latrach, R; Sassi, L; Darghouth, M A

    2012-08-01

    We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.

  14. Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

    PubMed

    Schwaiger, K; Wimmer, M; Huber-Schlenstedt, R; Fehlings, K; Hölzel, C S; Bauer, J

    2012-01-01

    A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis.

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

    SciTech Connect

    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.

  16. Oocyte development in bovine primordial follicles is promoted by activin and FSH within a two-step serum-free culture system.

    PubMed

    McLaughlin, Marie; Telfer, Evelyn E

    2010-06-01

    Quiescent follicles of large mammals initiate growth within cultured pieces of ovarian cortex. Systems capable of sustaining in vitro development from this early stage until oocyte maturation would allow investigation of mechanisms regulating oocyte development in its entirety. The aims of this study were 1) to determine whether bovine follicles initiated to grow in vitro could be isolated from the cortical environment, and could undergo further development and 2) to evaluate the effect of activin and FSH on the development of secondary follicles derived from primordial follicles. Fragments of bovine ovarian cortex were cultured in serum-free medium for 6 days; thereafter, secondary follicles were isolated for further culture. After a maximum total of 21 days in vitro, follicles were either processed for histological assessment or opened to release the oocyte-cumulus complexes for inspection by light microscopy. Compared with control, significant follicle and oocyte growth were observed in activin-exposed follicles, with or without FSH, with some oocyte diameters measuring over 100 microns following a total in vitro period of 15 days. Significant oestradiol secretion was observed in follicles cultured in activin alone after a total of 9 days in vitro compared with other treatment groups; however, this effect was not sustained. In summary, this study demonstrates the promotion of primordial bovine follicle development within a two-step serum-free culture system with oocyte diameters >100 mum achieved over 15 days in vitro. Further development of this system is needed to support complete oocyte growth and thereafter in vitro maturation.

  17. A rare adrenal incidentaloma: adrenal schwannoma.

    PubMed

    Adas, Mine; Ozulker, Filiz; Adas, Gokhan; Koc, Bora; Ozulker, Tamer; Sahin, Ilknur Mansuroglu

    2013-01-01

    Adrenal schwannoma is an extremely uncommon cause of incidentaloma. It originates from neural sheath Schwann cells of the adrenal gland. We report the case of a left adrenal schwannoma incidentally discovered in a 32-year-old woman during examination of bloated feeling and stomach ache. The patient was incidentally found to have a left adrenal mass of 9 cm on abdominal ultrasonography. Computed tomography (CT) of the abdomen and [(18)F] fluorodeoxyglucose positron emission tomography (PET) were also performed. Metabolic evaluation was unremarkable. Due to the large size of the tumor, left adrenalectomy was performed. The postoperative course was uneventful. Histological examination established the diagnosis of schwannoma. This diagnosis was supported by immunohistochemistry of S-100 and vimentin positivity. In conclusion, adrenal schwannoma is an extremely rare entity and can grow considerably in size. The present case report emphasizes that clinicians should be aware of the possibility of retroperitoneal schwannoma. Total excision of benign schwannoma is associated with a favorable outcome. To our knowledge, there are case reports of schwannoma with CT and magnetic resonance imaging findings in the literature, although this is the first schwannoma case with PET-CT imaging.

  18. Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

    PubMed

    Zhang, Jin-You; Wu, Yi; Zhao, Shuan; Liu, Zhen-Xing; Zeng, Shen-Ming; Zhang, Gui-Xue

    2015-09-15

    Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

  19. Evidence of bovine immunoglobulin G1 (IgG1) protease activity in partially purified culture supernate of Pasteurella haemolytica A1.

    PubMed Central

    Lee, C W; Shewen, P E

    1996-01-01

    In the bovine respiratory tract, IgG1 is a major secretory immunoglobulin (Ig), and both IgG1 and IgG2 are believed to be important in defense against pneumonic pasteurellosis (shipping fever) in calves. Here we provide evidence for hydrolysis of IgG1 in the presence of partially purified culture supernate (ppCS) from the respiratory pathogen Pasteurella haemolytica A1. Bovine IgG1 was hydrolysed sequentially into three distinct bands (approximately 39, 12, and 7 kDa respectively). Furthermore, partial hydrolysis of bovine IgG2 was observed, but neither bovine IgA nor IgM were affected by incubation with ppCS. These findings suggest that the production of an IgG1-specific protease by P. haemolytica A1 may be a virulence mechanism contributing to the pathogenesis of bovine pneumonic pasteurellosis. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. PMID:8785718

  20. Growth and development of rabbit oocytes in vitro: effect of fetal bovine serum concentration on culture medium.

    PubMed

    Sugimoto, H; Kida, Y; Miyamoto, Y; Kitada, K; Matsumoto, K; Saeki, K; Taniguchi, T; Hosoi, Y

    2012-09-15

    The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 μm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P < 0.1). The rate of development to the blastocyst stage was also higher in the medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P < 0.05). Next, using oocytes recovered from follicles 200 to 399 μm in diameter which were cultured in 0.05% FBS, oxygen consumption and the number of cells were analyzed. Blastocysts from oocytes grown in vitro with 0.05% FBS had reduced oxygen consumption and number of cells compared with those from ovulated oocytes (21.66 ± 4.54 × 10(14) vs. 50.19 ± 4.61 × 10(14) mol/sec, 244 ± 25 vs. 398 ± 24, P < 0.05). Rabbit oocytes grown in vitro with 0.05% FBS achieved pregnancy, but pregnancies were not maintained to term. In conclusion, the addition of 0.05% FBS to the culture medium for IVG improved developmental competence of rabbit oocytes grown in vitro.

  1. Effect of environmental particulates on cultured human and bovine endothelium. Cellular injury via an oxidant-dependent pathway

    SciTech Connect

    Garcia, J.G.; Dodson, R.F.; Callahan, K.S.

    1989-07-01

    The effects of respirable environmental fibers on cultures of human umbilical vein and bovine pulmonary artery endothelial cell monolayers were studied. Interaction among endothelial cell monolayers and amosite and chrysotile asbestos, attapulgite, fiberglass, or latex beads resulted in rapid phagocytosis of the particulates. A gradient of time-dependent and concentration-dependent endothelial cell injury (measured by specific 51Cr release) was observed with amosite and attapulgite being markedly toxic. Chrysotile and fiberglass were much less toxic, and latex beads were not significantly injurious at any time or dose examined. Responses of bovine pulmonary artery and human endothelial vein endothelial cells to fiber phagocytosis and fiber-induced injury were similar. In human umbilical cell monolayers, fiber-mediated stimulation of the arachidonate metabolite prostacyclin paralleled endothelial cell injury; i.e. amosite and attapulgite were stimulatory, whereas fiberglass (0-500 micrograms/ml) and latex beads (10(9) beads/ml) did not significantly increase prostacyclin generation. Although chrysotile was only weakly cytotoxic, significant stimulation of prostacyclin was observed at the highest dose tested (500 micrograms/ml). To investigate whether toxic oxygen species may be involved in fiber-induced cytotoxicity, oxidant scavengers or inhibitors were used in injury studies. Both superoxide dismutase (a scavenger of O2-) and catalase (an inhibitor of H2O2) produced significant protection against fiber-mediated endothelial cell injury. In addition, chelation by deferoxamine of elemental Fe present in the fiber preparations was also protective, suggesting Fe, via the modified Haber-Weiss reaction, may promote hydroxyl radical formation and contribute to endothelial cell injury induced by these particulates.

  2. Recovery of mitochondrial function and endogenous antioxidant systems in vitrified bovine oocytes during extended in vitro culture.

    PubMed

    Zhao, Xue-Ming; Du, Wei-Hua; Wang, Dong; Hao, Hai-Sheng; Liu, Yan; Qin, Tong; Zhu, Hua-Bin

    2011-12-01

    This study was designed to examine the recovery of mitochondrial function and endogenous antioxidant systems in vitrified oocytes during extended incubations. After 16 hr of in vitro maturation, bovine meiosis-II oocytes were vitrified, and then surviving oocytes were cultured an additional 8 hr. ATP content, ATP synthase activity, expression of ATP synthase F0 subunit 6 (ATP6) and 8 (ATP8) genes, and reactive oxygen species (ROS) levels were investigated in the vitrified oocytes during this additional period (4 or 8 hr). The results showed that: (1) the ATP content and ATP synthase activities in vitrified oocytes at 8 hr post-warming (754.6 fmol, 25.9 nmol NADH/min/mg) were significantly higher than in oocytes immediately warmed (568.3 fmol, 8.7 nmol NADH/min/mg), but still lower than in control oocytes (901.5 fmol, 30.7 nmol NADH/min/mg); (2) the relative expression of ATP6 and ATP8 was initially down-regulated in oocytes when they were first warmed, increased by 4 hr post-warming, and were again down-regulated by 8 hr post-warming; (3) ROS levels in oocytes at 0, 4, and 8 hr post-warming were significantly higher than in control oocytes; and (4) after parthenogenetic activation, the blastocyst rate of oocytes at 8 hr post-warming (26.7%) was significantly higher than that of oocytes immediately warmed (16.9%). These results indicated that mitochondrial function and endogenous antioxidant systems recovered significantly better in vitrified-thawed bovine oocytes with 8 hr of additional incubation, but they did not achieve the activity levels found in fresh oocytes.

  3. Osteopontin, osteocalcin and OB-cadherin expression in Synthetic nanohydroxyapatite vs bovine hydroxyapatite cultured Osteoblastic-like cells.

    PubMed

    Santarelli, A; Mascitti, M; Orsini, G; Memè, L; Rocchetti, R; Tiriduzzi, P; Sampalmieri, F; Putignano, A; Procaccini, M; Lo Muzio, L; Bambini, F

    2014-01-01

    Calcium phosphate ceramics have been applied in bone replacement for several decades due to their excellent biocompatibility, bioactivity, osteo-conductivity and mechanical strength. Several studies have demonstrated that porous hydroxyapatite (HA) is an excellent scaffold for osteogenic proliferation and differentiation of the osteoprogenitor cells. However, different methods of synthesis and production of HA ceramic-based materials may have considerable effect on the physical and biological properties. In the present work, two hydroxyapatite-based materials, a natural hydroxyapatite ceramic of bovine origin and a synthetic nano-cristalline hydroxyapatite were tested in vitro with MG63 cell line. The results displayed that both the materials demonstrated a good biocompatibility. The immunocytochemical stain revealed a different positivity of the osteogenic markers between the cultures with the biomaterials, and the control culture. Western blot data confirmed the immunocytochemical stain. Both the materials tested in the present study demonstrated a good biocompatibility with the osteoblastic cells allowing, at the same time, the osteogenic differentiation, and they may be useful in clinical use.

  4. Adrenal cortex ontogenesis.

    PubMed

    Lalli, Enzo

    2010-12-01

    During the early phases of development, adrenal glands share a common origin with kidneys and gonads. The action of diverse transcription factors, signalling pathways and endocrine signals is required for the individualization of the adrenal primordium and its subsequent differentiation into an adult adrenal gland, with massive remodelling taking place around the time of birth in humans. Here I summarize the most important steps by which the adrenal cortex is shaped and present an overview of the current understanding of the genes and molecular pathways implicated in adrenal development and involved in the pathogenesis of its congenital diseases. Evidence is accumulating that some pivotal factors acting during adrenocortical development also play an important role to regulate the growth of adrenocortical tumors, representing promising therapeutical targets for a biology-oriented therapy. 2010 Elsevier Ltd. All rights reserved.

  5. Hypertension and adrenal disorders.

    PubMed

    Blumenfeld, J D

    1993-03-01

    Abnormalities of adrenal cortical and medullary function are important causes of hypertension in adults. Mineralocorticoid hypertension, characterized by spontaneous hypokalemia with excessive kaliuresis and low plasma renin activity, is most commonly caused by aldosterone-producing adenoma or, less frequently, by nonadenomatous adrenal hyperplasia. However, recent evidence indicates that this classification oversimplifies the pathophysiologic diversity of this syndrome. Advances in steroid biochemistry and molecular biology have improved our ability to identify patients with various forms of mineralocorticoid hypertension and also provide evidence that they are underdiagnosed. Pheochromocytomas are most commonly located in the adrenal medulla, where they may overproduce norepinephrine or epinephrine. Appropriate screening of norepinephrine, epinephrine, and their metabolites is essential because tumors that secrete epinephrine exclusively may not present with hypertension and, thus, can be overlooked. Extra-adrenal pheochromocytomas are more prevalent than previously considered and pose special problems because they may be multicentric, difficult to locate, and more likely to be malignant than are adrenal pheochromocytomas.

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

    PubMed Central

    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

  7. Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines.

    PubMed

    Chen, Di; Xin, Xiao-Xuan; Qian, Hao-Cheng; Yu, Zhang-Yin; Shen, Li-Rong

    2016-06-01

    Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%‒56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

  8. Isolation, in vitro culture and identification of a new type of mesenchymal stem cell derived from fetal bovine lung tissues.

    PubMed

    Hu, Pengfei; Pu, Yabin; Li, Xiayun; Zhu, Zhiqiang; Zhao, Yuhua; Guan, Weijun; Ma, Yuehui

    2015-09-01

    Lung‑derived mesenchymal stem cells (LMSCs) are considered to be important in lung tissue repair and regenerative processes. However, the biological characteristics and differentiation potential of LMSCs remain to be elucidated. In the present study, fetal lung‑derived mesenchymal stem cells (FLMSCs) were isolated from fetal bovine lung tissues by collagenase digestion. The in vitro culture conditions were optimized and stabilized and the self‑renewal ability and differentiation potential were evaluated. The results demonstrated that the FLMSCs were morphologically consistent with fibroblasts, were able to be cultured and passaged for at least 33 passages and the cell morphology and proliferative ability were stable during the first 10 passages. In addition, FLMSCs were found to express CD29, CD44, CD73 and CD166, however, they did not express hematopoietic cell specific markers, including CD34, CD45 and BOLA‑DRα. The growth kinetics of FLMSCs consisted of a lag phase, a logarithmic phase and a plateau phase, and as the passages increased, the proliferative ability of cells gradually decreased. The majority of FLMSCs were in G0/G1 phase. Following osteogenic induction, FLMSCs were positive for the expression of osteopontin and collagen type I α2. Following neurogenic differentiation, the cells were morphologically consistent with neuronal cells and positive for microtubule‑associated protein 2 and nestin expression. It was concluded that the isolated FLMSCs exhibited typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their proliferation and the maintenance of stemness. The present study illustrated the potential application of lung tissue as an adult stem cell source for regenerative therapies.

  9. Effect of Removal of Spermatogonial Stem Cells (SSCs) from In Vitro Culture on Gene Expression of Niche Factors in Bovine

    PubMed Central

    Akbarinejad, Vahid; Tajik, Parviz; Movahedin, Mansoureh; Youssefi, Reza

    2016-01-01

    Background: Niche cells, regulating Spermatogonial Stem Cells (SSCs) fate are believed to have a reciprocal communication with SSCs. The present study was conducted to evaluate the effect of SSC elimination on the gene expression of Glial cell line-Derived Neurotrophic Factor (GDNF), Fibroblast Growth Factor 2 (FGF2) and Kit Ligand (KITLG), which are the main growth factors regulating SSCs development and secreted by niche cells, primarily Sertoli cells. Methods: Following isolation, bovine testicular cells were cultured for 12 days on extracellular matrix-coated plates. In the germ cell-removed group, the SSCs were removed from the in vitro culture using differential plating; however, in the control group, no intervention in the culture was performed. Colony formation of SSCs was evaluated using an inverted microscope. The gene expression of growth factors and spermatogonia markers were assessed using quantitative real time PCR. Results: SSCs colonies were developed in the control group but they were rarely observed in the germ cell-removed group; moreover, the expression of spermatogonia markers was detected in the control group while it was not observed in the germ cell-removed group, substantiating the success of SSCs removal. The expression of Gdnf and Fgf2 was greater in the germ cell-removed than control group (p<0.05), whereas the expression of Kitlg was lower in the germ cell-removed than control group (p< 0.05). Conclusion: In conclusion, the results revealed that niche cells respond to SSCs removal by upregulation of GDNF and FGF2, and downregulation of KITLG in order to stimulate self-renewal and arrest differentiation. PMID:27563426

  10. The relation of RNA synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

    PubMed Central

    McQuillan, D J; Handley, C J; Robinson, H C; Ng, K; Tzaicos, C

    1986-01-01

    Addition of actinomycin D (or cordycepin, an alternative inhibitor of RNA synthesis) to cartilage cultures resulted in a first-order decrease in the rate of incorporation of [35S]sulphate into proteoglycan (half-life = 7.5 +/- 1.1 h). Addition of 1.0 mM-benzyl beta-D-xyloside relieved the initial inhibition of glycosaminoglycan synthesis induced by actinomycin D; however, after a lag of about 10 h the rate of xyloside-initiated glycosaminoglycan synthesis also decreased with apparent first-order kinetics (half-life = 7.1 +/- 1.8 h), which paralleled the decrease in the rate of core-protein-initiated glycosaminoglycan synthesis. The hydrodynamic size of the proteoglycans formed in the presence of actinomycin D remained essentially constant (Kav. 0.21-0.23), whereas the constituent glycosaminoglycan chains were larger than those formed by control cultures, which suggested that the core protein was substituted with fewer but larger glycosaminoglycan chains. Proteoglycans formed in the presence of beta-D-xyloside were significantly smaller (Kav. approximately 0.33) than those synthesized by control cultures, and were further diminished in size after exposure of cultures to actinomycin D. Glycosaminoglycan chains synthesized by these same cultures on to both core-protein and xyloside acceptors were also smaller than those of control cultures. The decrease in synthesis observed after exposure to actinomycin D was not reflected by any significant decrease in the activities of several glycosyltransferases involved in chondroitin sulphate synthesis (galactosyltransferase-I, galactosyltransferase-II, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II). PMID:2427073

  11. Effect of extracellular matrix on testosterone production during in vitro culture of bovine testicular cells.

    PubMed

    Akbarinejad, Vahid; Tajik, Parviz; Movahedin, Mansoureh; Youssefi, Reza

    2017-01-01

    Testosterone is believed to play a significant role in spermatogenesis, but its contribution to the process of spermatogenesis is not completely understood. Given that extracellular matrix (ECM) facilitates differentiation of spermatogonial stem cells (SSCs) during culture, the present study was conducted to elucidate whether testosterone contribute to the permissive effect of ECM on SSCs differentiation. In experiment 1, testosterone production was measured in testicular cells cultured for 12 days on ECM or plastic (control). In experiment 2, testosterone production was assessed in testicular cells cultured on ECM or plastic (control) and exposed to different concentrations of hCG. In experiment 3, the gene expression of factors involved in testosterone production was analyzed. Testosterone concentration was lower in ECM than in the control group in experiment 1 (p < 0.05). In experiment 2, testosterone concentration was increased in response to hCG in both groups but cells cultured on ECM were more responsive to hCG than those cultured on plastic (p < 0.05). In the experiment 3, qRT-PCR revealed the inhibitory effect of ECM on the gene expression of steroidogenic acute regulatory protein (StAR) (p < 0.05). Nevertheless, the expression of LH receptor was greater in ECM-exposed than in unexposed cells (p < 0.05). In conclusion, the present study showed that inhibiting the expression of StAR, ECM could lower testosterone production by Leydig cells during in vitro culture. In addition, the results indicated that ECM could augment the responsiveness of Leydig cells to hCG through stimulating the expression of LH receptor.

  12. Effect of C-reactive protein on Fcgamma receptor II in cultured bovine endothelial cells.

    PubMed

    Escribano-Burgos, Marta; López-Farré, Antonio; del Mar González, María; Macaya, Carlos; García-Méndez, Antonio; Mateos-Cáceres, Petra J; Alonso-Orgaz, Sergio; Carrasco, Carolina; Rico, Luis A; Porres Cubero, Juan Carlos

    2005-01-01

    The major CRP (C-reactive protein) receptor on leucocytes has been identified as the low-affinity IgG receptor Fcgamma receptor II (CD32). Our aim was to assess whether inflammation may modify the presence of the CD32 receptor in BAEC (bovine aortic endothelial cells). Confocal microscopy experiments showed a weak expression of the CD32 receptor in control BAEC that was slightly increased by 10 microg/ml CRP. Incubation of BAEC with TNF-alpha (tumour necrosis factor-alpha) did not modify the fluorescence signal of CD32. Addition of CRP to TNF-alpha-incubated BAEC enhanced the fluorescence signal of the CD32 receptors. The CD32 receptors showed a perinuclear cytoplasmic localization in BAEC. An alteration of the NO (nitric oxide)-dependent vasorelaxation has been defined as endothelial dysfunction. Endothelial dysfunction has been associated with the presence of superoxide anion and with a reduction in the expression of the eNOS (endothelial NO synthase). A concentration of CRP similar to that detected in patients with cardiovascular risk (10 microg/ml) failed to modify the generation of superoxide anion stimulated by TNF-alpha. Western blot experiments showed that TNF-alpha decreased the expression of the eNOS protein, which was partially protected by treatment with 10 microg/ml CRP. The protective effect of 10 microg/ml CRP on eNOS expression in TNF-alpha-incubated BAEC was prevented by an antibody against CD32 receptors. In conclusion, the present results suggest that, although CRP has been associated with inflammation, CRP may protect the expression of eNOS protein against pro-inflammatory mediators such as TNF-alpha.

  13. Purification and physiochemical properties of a recombinant bovine growth hormone produced by cultured murine fibroblasts.

    PubMed

    Leung, F C; Jones, B; Steelman, S L; Rosenblum, C I; Kopchick, J J

    1986-10-01

    Mouse fibroblast cell lines which secrete bovine (b) GH have been generated. This was accomplished by cotransforming mouse L cells (thymidine kinase-negative [TK-] and adenine phosphoribosyl transferase-negative [APRT-]) with DNA molecules encoding the Rous sarcoma virus-long-terminal repeat and bGH genes along with the herpes viral TK gene and the hamster APRT gene. One stable cell line, Pd lambda-pbGH 4-13, was found to secrete approximately 75 micrograms bGH per 24 h/5.0 X 10(6) cells. Media from this cell line were collected for purification of recombinant bGH (rbGH). Purification involved (NH4)2SO4 fractionation, ion-exchange chromatography, and gel filtration on Sephacryl S-200. The rbGH was characterized by bioassay, RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis. Results of these analyses were compared with those obtained with a highly purified pituitary bGH. In the rat tibia bioassay, rbGH was found to be as potent as pituitary bGH. Results from the RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis and Western blot analysis also suggested that the rbGH was similar to that of pituitary origin. Amino acid composition, partial (amino-terminal) sequence, and tryptic peptide maps were also found to be similar between the rbGH and pituitary bGH preparations. The amino terminus of the rbGH showed similar heterogeneity to that of the bGH of pituitary origin. We conclude that rbGH which was synthesized, processed, and secreted from transformed mouse fibroblasts possessed almost exactly the same physiochemical properties as pituitary bGH.

  14. 78 SUPPLEMENTATION WITH CARNOSINE DURING IN VITRO CULTURE IMPROVES THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS.

    PubMed

    Le Bourhis, D; Verachten, M; Salvetti, P; Hochet, M; Schibler, L

    2016-01-01

    The objective of the present study was to determine the effect of supplementation of culture medium with carnosine (β-alanyl-l-histidine; Sigma, St-Quentin Fallavier, France), a reactive oxygen species scavenger, on in vitro bovine embryo development and survival following cryopreservation. Abattoir-derived bovine oocytes (4 replicates) were in vitro matured and fertilized with frozen-thawed semen of one bull, according to our standard procedures. In Experiment 1, 20h after IVF, groups of presumptive zygotes were cultured in 30μL of SOF BSAaa+1% oestrus cow serum with 0 (control; n=205) or 5μgmL(-1) of carnosine (n=209) under humidified air with 5% CO2, 5% O2, and 88% N2. Cleavage rates were determined on Day 2, and the blastocyst rates and grade were assessed on Day 7 according to IETS classification. Day 7 grade 1 expanded blastocysts (n=25 control and n=27 carnosine) were frozen in 1.5M ethylene glycol+0.1M sucrose. Embryos were thawed and then cultured for 72h in SOF-BSAaa+1% oestrus cow serum for re-expansion and hatching rate assessments at +24h, +48h, and +72h post-thawing. In Experiment 2, presumed zygotes were cultured in SOF BSAaa+1% oestrus cow serum with 0 (control; n=48) or 5μgmL(-1) of carnosine (n=48) in a WOW dish and observed with Time Laps Cinematography (Primo Vision®, VitroLife, Göteborg, Sweden). Images were recorded every 15min for up to 168h post-insemination. For embryos that reached the blastocyst stage, mean timing of the first cleavage (C1; 2-cell stage), second cleavage (C2; 4-cell stage), second cleavage to compaction (C3), and blastocoel cavity appearance (B4) were recorded. Chi-square test for Experiment 1 and Student's t-test for Experiment 2 were used, and differences were considered significant at P<0.05. In Experiment 1, no differences were observed in cleavage rate, blastocyst rate on Day 7, and grade 1 blastocyst rate between both control and carnosine groups (84.0±4.2v.85.2±3.8, P=0.7; 46.9±7.1v. 45.0±7.5, P=0.7; 24

  15. Effect of embryo density on in vitro development and gene expression in bovine in vitro-fertilized embryos cultured in a microwell system.

    PubMed

    Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Aikawa, Yoshio; Ohtake, Masaki; Matsuda, Hideo; Kobayashi, Shuji; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

    2013-01-01

    To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

  16. Effect of Embryo Density on In Vitro Development and Gene Expression in Bovine In Vitro-fertilized Embryos Cultured in a Microwell System

    PubMed Central

    SUGIMURA, Satoshi; AKAI, Tomonori; HASHIYADA, Yutaka; AIKAWA, Yoshio; OHTAKE, Masaki; MATSUDA, Hideo; KOBAYASHI, Shuji; KOBAYASHI, Eiji; KONISHI, Kazuyuki; IMAI, Kei

    2012-01-01

    Abstract To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density. PMID:23154384

  17. Management of Adrenal Masses.

    PubMed

    Bhat, Hattangadi Sanjay; Tiyadath, Balagopal Nair

    2017-03-01

    An adrenal mass can be either symptomatic or asymptomatic in the form of adrenal incidentalomas (AIs) in up to 8 % in autopsy and 4 % in imaging series. Once a diagnosis of adrenal mass is made, we need to differentiate whether it is functioning or nonfunctioning, benign, or malignant. In this article, we provide a literature review of the diagnostic workup including biochemical evaluation and imaging characteristics of the different pathologies. We also discuss the surgical strategies with laparoscopy as the mainstay with partial adrenalectomy in select cases and adrenalectomy in large masses. Follow-up protocol of AIs and adrenocortical carcinoma is also discussed.

  18. Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-{gamma}

    SciTech Connect

    Kanata, Sohya; Akagi, Masao . E-mail: makagi@med.kindai.ac.jp; Nishimura, Shunji; Hayakawa, Sumio; Yoshida, Kohji; Sawamura, Tatsuya; Munakata, Hiroshi; Hamanishi, Chiaki

    2006-09-29

    It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-{gamma} was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-{gamma} inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-{gamma} and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.

  19. Bovine serum albumin contained in culture medium used in artificial insemination is an important anaphylaxis risk factor.

    PubMed

    Pagán, Juan A; Postigo, Idoia; Rodríguez-Pacheco, Jorge R; Peña, Maribel; Guisantes, Jorge A; Martínez, Jorge

    2008-11-01

    To analyze the cause of the anaphylactic reaction after a standard artificial insemination process in a patient diagnosed with asthma. Case report. Residencia Sanitaria Virgen de la Arrixaca (Murcia, Spain) and University of the Basque Country (Vitoria, Spain). A 30-year-old woman with a previous medical history compatible with respiratory allergy who suffered an anaphylactic reaction after an artificial insemination with spermatozoids in capable medium (Upgraded B2 INRA medium; Laboratories CCD, Paris, France). Cutaneous tests and specific IgE levels to inhalant allergens, grass and Olea pollens, and insemination medium were performed. Specific IgE levels to mammal epithelia and bovine serum albumin (BSA). Skin prick tests were positive for inhalant allergens such as mites, cat, dog, horse, and rabbit epithelia, grasses and Olea pollens, and the insemination medium. The beta-lactamic tests were negative. The determination of specific IgE demonstrated positive values to mammal epithelia and mammal serum albumins including BSA. We report a case of an anaphylactic reaction to the BSA included in the insemination culture medium induced by a subclinical sensitivity to serum albumins of mammal epithelia. A previous testing with the medium is recommended and specific testing might be needed in women who have a history of animal epithelium allergies.

  20. Inhibition of cultured bovine aortic endothelial cell proliferation by sodium spirulan, a new sulfated polysaccharide isolated from Spirulina platensis.

    PubMed

    Kaji, Toshiyuki; Fujiwara, Yasuyuki; Hamada, Chieko; Yamamoto, Chika; Shimada, Satomi; Lee, Jung-Bum; Hayashi, Toshimitsu

    2002-06-01

    Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, which consists of two types of disaccharide repeating units, O-hexuronosyl-rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methylrhamnose (acofriose) with sulfate groups, other minor saccharides and sodium ion. Vascular endothelial cells are present on the inner surface of blood vessels in a monolayer and have anticoagulant properties. To address the question whether Na-SP influences the maintenance of endothelial cell monolayers, we investigated the proliferation of cultured bovine aortic endothelial cells treated with Na-SP. It was found that Na-SP has an inhibitory activity on endothelial cell proliferation accompanied with suppression of whole protein synthesis but without non-specific cell damage. The inhibitory activity of Na-SP was the strongest when compared to that of heparan sulfate, heparin, dextran sulfate, dermatan sulfate, chondroitin sulfate A/C and hyaluronan. Furthermore, it was shown that the inhibitory activity of Na-SP disappeared by either desulfation or depolymerization. The present data suggest that Na-SP is a unique sulfated polysaccharide that strongly inhibits vascular endothelial cell proliferation, and the inhibitory activity requires polymerization of sulfated O-rhamnosyl-acofriose repeating units.

  1. Oleic acid induces specific alterations in the morphology, gene expression and steroid hormone production of cultured bovine granulosa cells.

    PubMed

    Yenuganti, Vengala Rao; Viergutz, Torsten; Vanselow, Jens

    2016-06-01

    After parturition, one of the major problems related to nutritional management that is faced by the majority of dairy cows is negative energy balance (NEB). During NEB, excessive lipid mobilization takes place and hence the levels of free fatty acids, among them oleic acid, increase in the blood, but also in the follicular fluid. This accumulation can be associated with serious metabolic and reproductive disorders. In the present study, we analyzed the effects of physiological concentrations of oleic acid on cell morphology, apoptosis, necrosis, proliferation and steroid production, and on the abundance of selected transcripts in cultured bovine granulosa cells. Increasing oleic acid concentrations induced intracellular lipid droplet accumulation, thus resulting in a foam cell-like morphology, but had no effects on apoptosis, necrosis or proliferation. Oleic acid also significantly reduced the transcript abundance of the gonadotropin hormone receptors, FSHR and LHCGR, steroidogenic genes STAR, CYP11A1, HSD3B1 and CYP19A1, the cell cycle regulator CCND2, but not of the proliferation marker PCNA. In addition, treatment increased the transcript levels of the fatty acid transporters CD36 and SLC27A1, and decreased the production of 17-beta-estradiol and progesterone. From these data it can be concluded that oleic acid specifically affects morphological and physiological features and gene expression levels thus altering the functionality of granulosa cells. Suggestively, these effects might be partly due to the reduced expression of FSHR and thus the reduced responsiveness to FSH stimulation.

  2. Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.

    PubMed

    Naaijkens, B A; Niessen, H W M; Prins, H-J; Krijnen, P A J; Kokhuis, T J A; de Jong, N; van Hinsbergh, V W M; Kamp, O; Helder, M N; Musters, R J P; van Dijk, A; Juffermans, L J M

    2012-04-01

    Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

  3. Hepatoma-derived growth factor: from the bovine uterus to the in vitro embryo culture.

    PubMed

    Gómez, E; Correia-Álvarez, E; Caamaño, J N; Díez, C; Carrocera, S; Peynot, N; Martín, D; Giraud-Delville, C; Duranthon, V; Sandra, O; Muñoz, M

    2014-10-01

    Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

  4. Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions.

    PubMed

    Gad, Ahmed; Hoelker, Michael; Besenfelder, Urban; Havlicek, Vitezslav; Cinar, Ulas; Rings, Franca; Held, Eva; Dufort, Isabelle; Sirard, Marc-André; Schellander, Karl; Tesfaye, Dawit

    2012-10-01

    Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

  5. Induction by Glucocorticoids of Angiotensin Converting Enzyme Production from Bovine Endothelial Cells in Culture and Rat Lung In Vivo

    PubMed Central

    Mendelsohn, F. A. O.; Lloyd, C. J.; Kachel, C.; Funder, J. W.

    1982-01-01

    The effect of corticosteroids on angiotensin converting enzyme was investigated in endothelial cell cultures and intact rat lung. Cultured endothelial cells from bovine aorta showed net production of angiotensin converting enzyme (ACE) over 2 d culture in serum-free medium. Dexamethasone (DM) increased cell ACE activity six- to sevenfold at 100 nM with a threshold effect at 0.3 nM. The effect of DM on ACE production was completely inhibited by actinomycin D or cycloheximide. Deoxycorticosterone (DOC) and aldosterone were markedly less active, with a threshold near 100 nM and significant (two to threefold) stimulation of ACE activity at 1 μM. In cells incubated in the presence of 10 nM DM, DOC (10 μM) significantly inhibited ACE production compared with 10 nM DM alone, suggesting that DOC is a partial agonist/partial antagonist in this enzyme system. Protein content of cells or medium was unchanged by steroids at all doses used. In vivo, adrenalectomized rats showed lower pulmonary ACE compared with intact controls, and when injected with DM (40 μg/d for 4 d) showed a significant (twofold, P < 0.002) increase in lung ACE over oil-injected, adrenalectomized controls; serum ACE did not change. Injection with DOC (40 μg/d) or aldosterone (10 μg/d) had no effect on lung or serum ACE. Over a range (0.6 to 2,000 μg) of concentrations of DM administered daily for 7 d, the dose-response curve of DM for induction of pulmonary ACE mirrored that for thymolysis; for both, half-maximal effects were seen at ∼6 μg DM/d, and plateau levels at 60 μg/d. We conclude that glucocorticoids are potent inducers of ACE activity in endothelial cells in culture and in rat lung in vivo, and that the action of aldosterone and DOC reflects occupancy of glucocorticoid receptors. This effect may be of (patho)physiological relevance in regulating levels of ACE in local vascular beds, and thereby modulating local levels of the vasoactive peptides angiotensin II and bradykinin. PMID:6286730

  6. Cytotoxicity of local anesthetics and nonionic contrast agents on bovine intervertebral disc cells cultured in a three-dimensional culture system.

    PubMed

    Chee, Ana V; Ren, Jing; Lenart, Brett A; Chen, Er-Yun; Zhang, Yejia; An, Howard S

    2014-03-01

    Carragee et al. reported an accelerated progression of lumbar intervertebral disc (IVD) degeneration after discography in a human trial. Local anesthetics and contrast agents have exhibited toxicity to cardiac, renal, and neuronal cells. We hypothesize that local anesthetics or contrast agents commonly injected into the disc space during discography may result in cytotoxicity in vitro. In this study, we compared the cytotoxicity of these agents, alone or in combination, using nucleus pulposus (NP) and annulus fibrosus (AF) cells in a three-dimensional (3D) culture system. The purpose of this study was to examine the effects of local anesthetics and contrast agents on IVD cells to help guide their usage in future clinical practices. Ours was an in vitro study to assess the cytotoxicity of local anesthetics and contrast agents commonly used in discography, using bovine NP and AF cells cultured in a 3D system. Bovine NP and AF cells were isolated and encapsulated in alginate beads and cultured in media completed with serum and ascorbic acid. Beads were transferred to a 24-well plate and treated with local anesthetics, nonionic contrast agents, or with saline as a control for 2, 6, and 16 hours. Three different concentrations of local anesthetics, lidocaine and bupivacaine, were tested: 0.25%, 0.125%, and 0.0625%. Two different dilutions (1:2 or 1:4) of nonionic contras agents, iohexol and iopamidol, were tested. In a parallel study, beads were incubated with a combination of local anesthetics at equipotent concentrations and contrast agents for 6 hours. Cells were then examined with the LIVE/DEAD cell assay. Live cells (fluorescing green) and dead cells (fluorescing red) were visualized using fluorescent microscopy. The percentage of live cells after treatment was determined. More cell death was observed when NP and AF cells were incubated with anesthetics than contrast agents at the concentrations tested. When tested at equipotent concentrations, 0

  7. Managing Adrenal Insufficiency

    MedlinePlus

    ... the body. • Surgical removal of the adrenals Temporary AI is caused by some medications, infections, and/or surgeries. Causes of temporary AI include the following: • Transsphenoidal surgery for Cushing’s disease ...

  8. Adrenal gland and bone.

    PubMed

    Hardy, Rowan; Cooper, Mark S

    2010-11-01

    The adrenal gland synthesizes steroid hormones from the adrenal cortex and catecholamines from the adrenal medulla. Both cortisol and adrenal androgens can have powerful effects on bone. The overproduction of cortisol in Cushing's disease leads to a dramatic reduction in bone density and an increase risk of fracture. Overproduction of adrenal androgens in congenital adrenal hyperplasia (CAH) leads to marked changes in bone growth and development with early growth acceleration but ultimately a significant reduction in final adult height. The role of more physiological levels of glucocorticoids and androgens on bone metabolism is less clear. Cortisol levels measured in elderly individuals show a weak correlation with measures of bone density and change in bone density over time with a high cortisol level associated with lower bone density and more rapid bone loss. Cortisol levels and the dynamics of cortisol secretion change with age which could also explain some age related changes in bone physiology. It is also now clear that adrenal steroids can be metabolized within bone tissue itself. Local synthesis of cortisol within bone from its inactive precursor cortisone has been demonstrated and the amount of cortisol produced within osteoblasts appears to increase with age. With regard to adrenal androgens there is a dramatic reduction in levels with aging and several studies have examined the impact that restoration of these levels back to those seen in younger individuals has on bone health. Most of these studies show small positive effects in women, not men, but the skeletal sites where benefits are seen varies from study to study.

  9. Immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation.

    PubMed

    Weng, Qiang; Tanaka, Yumiko; Taniyama, Hiroyuki; Tsunoda, Nobuo; Nambo, Yasuo; Watanabe, Gen; Taya, Kazuyoshi

    2007-10-01

    To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.

  10. In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

    PubMed

    Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

    2010-02-18

    The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

  11. Comparison of colony-formation efficiency of bovine fetal fibroblast cell lines cultured with low oxygen, hydrocortisone, L-carnosine, bFGF, or different levels of FBS.

    PubMed

    Talbot, Neil C; Powell, Anne M; Caperna, Thomas J

    2004-01-01

    A comparison of colony-formation efficiency (CFE) was made between six independent bovine fetal fibroblast (BFF) cell lines used in somatic cell nuclear transfer. Variation in CFE was assessed under different culture conditions. The conditions examined were ambient atmosphere (approximately 20% oxygen) culture versus 5% oxygen culture, three levels of fetal bovine serum (FBS) in the medium (5%, 10% or 20%), and the amendment of 10% FBS medium with basic fibroblast growth factor (1 ng/mL), L-carnosine (20 mM), or hydrocortisone (1 microM). The six BFF cell lines showed significant differences from one another in CFE. No significant difference in CFE was found with reduced oxygen culture. L-Carnosine also had no significant effect on CFE. A FBS concentration of 10% was found to produce the best overall CFE. Hydrocortisone treatment reduced the size of colonies although the number of colonies formed was not affected. Basic FGF increased the size of colonies but the number of colonies formed was not affected. The results showed that different BFF cell lines varied significantly in their CFE. Also, some medium supplements or culture conditions that have shown positive CFE effects on the fibroblasts of other species failed to show significant positive CFE effects on the BFF cell lines tested.

  12. Effect of cell shrinkage on permeability of cultured bovine aortic endothelia and frog mesenteric capillaries.

    PubMed Central

    Kajimura, M; O'Donnell, M E; Curry, F E

    1997-01-01

    1. We have tested the hypothesis that a reduction in endothelial cell volume increases microvessel permeability and that the degree of endothelial cell attachment to their basement membranes determines the magnitude of permeability changes caused by a reduction in endothelial cell volume. 2. A decrease in endothelial cell volume was imposed on both intact microvessels and cultured endothelial monolayers by raising osmolarity by 100 mosmol l-1. 3. We found that hypertonic solutions did not increase the hydraulic permeability (Lp) of individually perfused venular microvessels in frog mesentery when the perfusate contained albumin. Hypertonic solutions did increase Lp, however, after we perfused the microvessels with the peptide Gly-Arg-Gly Asp-Thr-Pro (GRGDTP; 0.3 mmol l-1), to disrupt integrin-dependent endothelial cell (EC) attachment to the extracellular matrix (ECM). 4. After albumin was removed from the perfusate, hypertonic solutions increased Lp of microvessels and the permeability of endothelial monolayers to alpha-lactalbumin. 5. Our findings indicate that endothelial cell integrin-ECM binding plays a role in transducing changes in cell volume and/or shape into changes in permeability. We hypothesize that removal of albumin from the vascular perfusate may compromise EC-ECM interactions via an integrin-dependent mechanism. PMID:9306282

  13. Interaction between cadmium and zinc in the production and sulfation of glycosaminoglycans in cultured bovine vascular endothelial cells.

    PubMed

    Ohkawara, S; Kaji, T; Yamamoto, C; Fujiwara, Y; Sakamoto, M; Kozuka, H

    1996-02-09

    Previously, we showed that cadmium stimulates the production of glycosaminoglycans (GAGs) but inhibits their sulfation in cultured bovine aortic endothelial cells. The effect of zinc on such alterations of GAGs induced by cadmium was investigated in the present study. The incorporation of [3H]glucosamine and [35S]sulfate into GAGs was determined by the cetylpyridinium chloride precipitation method as a marker of GAG production and GAG sulfation, respectively. The incorporation of both [3H]glucosamine and [35S]sulfate was not changed in GAGs accumulated in the endothelial cell layer and the conditioned medium after exposure to zinc at 20 micrometers or less alone. A simultaneous exposure of the endothelial cell layer to zinc at 20 micrometers or less and cadmium at 2 micrometers resulted in prevention of the cadmium-induced decrease in [35S]sulfate incorporation; however, the cadmium-induced increase in [3H]glucosamine incorporation was not affected by zinc. Characterization of GAGs in the cell layer revealed that such an interaction between zinc and cadmium occurred in both heparan sulfate and the other GAGs. Zinc significantly prevented the inhibition of either [3H]thymidine or [3H]leucine incorporation caused by cadmium with less accumulation of intracellular cadmium suggesting that zinc decreased intracellular cadmium and protected endothelial cells from cadmium-induced inhibition of DNA and protein synthesis. The present data showed that a simultaneous exposure to cadmium and zinc resulted in an increase in heparan sulfate without a reduction of sulfation in the endothelial cell layer. The alteration may potentiate the antihrombogenic property of vascular endothelium.

  14. Interaction between cadmium and zinc in the production and sulfation of glycosaminoglycans in cultured bovine vascular endothelial cells

    SciTech Connect

    Ohkawara, Susumu; Kaji, Toshiyuki; Yamamoto, Chika

    1996-02-09

    Previously, we showed that cadmium stimulates the production of glycosaminoglycans (GAGs) but inhibits their sulfation in cultured bovine aortic endothelial cells. The effect of zinc on such alterations of GAGs induced by cadmium was investigated in the present study. The incorporation of [{sup 3}H]glucosamine and [{sup 35}S]sulfate into GAGs was determined by the cetylpyridinium chloride precipitation method as a marker of GAG production and GAG sulfation, respectively. The incorporation of both [{sup 3}H]glucosamine and [{sup 35}S]sulfate was not changed in GAGs accumulated in the endothelial cell layer and the conditioned medium after exposure to zinc at 20 {mu}M or less alone. A simultaneous exposure of the endothelial cell layer to zinc at 20 {mu}M or less and cadmium at 2{mu}M resulted in prevention of the cadmium-induced decrease in [{sup 35}S]sulfate incorporation; however, the cadmium-induced increase in [{sup 3}H]glucosamine incorporation was not affected by zinc. Characterization of GAGs in the cell layer revealed that such an interaction between zinc and cadmium occurred in both heparan sulfate and the other GAGs. Zinc significantly prevented the inhibition of either [{sup 3}H]thymidine or [{sup 3}H]leucine incorporation caused by cadmium with cadmium and protected endothelial cells from cadmium-induced inhibition of DNA and protein synthesis. The present data showed that a simultaneous exposure to cadmium and zinc resulted in an increase in heparan sulfate without a reduction of sulfation in the endothelial cell layer. The alteration may potentiate the antithrombogenic property of vascular endothelium. 30 refs., 2 figs., 3 tabs.

  15. Preferential adsorption of fetal bovine serum on bare and aromatic thiol-functionalized gold surfaces in cell culture media.

    PubMed

    Park, Jin; Park, Jin-Ho; Ock, Kwang-Su; Ganbold, Erdene-Ochir; Song, Nam Woong; Cho, Keunchang; Lee, So Yeong; Joo, Sang-Woo

    2011-11-01

    Intracellular uptake of serum-coated gold nanoparticles (AuNPs) in a single mammalian cell was examined in order to investigate the interactions of cell culture media and aromatic thiol-functionalized gold surfaces using micro-spectroscopic tools. The AuNPs modified by the aromatic thiols of para-aminobenzenethiol (ABT), para-hydroxy benzenethiol (HBT), and para-carboxylic benzenethiol (CBT, para-mercaptobenzoic acid) bearing NH(2), OH, and COOH surface functional groups are presumed to adsorb the serum proteins as indicated from the compiled quartz crystal microbalance (QCM) data. The QCM results indicate that among the constituents, fetal bovine serum (FBS) should be the major adsorbate species on AuNPs incubated in Roswell Park Memorial Institute (RPMI) medium. The functionalized AuNPs were found to be internalized as an aggregation state in mammalian cells as evidenced by transmission electron microscopy (TEM) images. We monitored such cellular uptake behaviors of aromatic thiol-modified AuNPs using dark-field microscopy (DFM)-guided confocal surface-enhanced Raman scattering techniques in order to identify the three-dimensional localization inside the single cell. We found that the uptake amounts of ABT, HBT, and CBT were similar by counting up to 70 particles inside the cells incubated in the solution mixture of the aromatic thiol and 1,4-phenylenediisocyanide (PDIC) as a reference. This result indicates for the short aromatic thiol compounds, the AuNPs should enter the cell after the serum-coating regardless of the surface functional groups. Considering that the aromatic thiols have little effect on the serum coating, the DFM/SERS method is an effective tool for monitoring the localization of AuNPs inside a single cell. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Interleukin-6-mediated signaling in adrenal medullary chromaffin cells.

    PubMed

    Jenkins, Danielle E; Sreenivasan, Dharshini; Carman, Fiona; Samal, Babru; Eiden, Lee E; Bunn, Stephen J

    2016-12-01

    The pro-inflammatory cytokines, tumor necrosis factor-α, and interleukin-1β/α modulate catecholamine secretion, and long-term gene regulation, in chromaffin cells of the adrenal medulla. Since interleukin-6 (IL6) also plays a key integrative role during inflammation, we have examined its ability to affect both tyrosine hydroxylase activity and adrenomedullary gene transcription in cultured bovine chromaffin cells. IL6 caused acute tyrosine/threonine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and serine/tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3). Consistent with ERK1/2 activation, IL6 rapidly increased tyrosine hydroxylase phosphorylation (serine-31) and activity, as well as up-regulated genes, encoding secreted proteins including galanin, vasoactive intestinal peptide, gastrin-releasing peptide, and parathyroid hormone-like hormone. The effects of IL6 on the entire bovine chromaffin cell transcriptome were compared to those generated by G-protein-coupled receptor (GPCR) agonists (histamine and pituitary adenylate cyclase-activating polypeptide) and the cytokine receptor agonists (interferon-α and tumor necrosis factor-α). Of 90 genes up-regulated by IL6, only 16 are known targets of IL6 in the immune system. Those remaining likely represent a combination of novel IL6/STAT3 targets, ERK1/2 targets and, potentially, IL6-dependent genes activated by IL6-induced transcription factors, such as hypoxia-inducible factor 1α. Notably, genes induced by IL6 include both neuroendocrine-specific genes activated by GPCR agonists, and transcripts also activated by the cytokines. These results suggest an integrative role for IL6 in the fine-tuning of the chromaffin cell response to a wide range of physiological and paraphysiological stressors, particularly when immune and endocrine stimuli converge. © 2016 International Society for Neurochemistry.

  17. Low oxygen tension and relative defined culture medium with 3, 4-dihydroxyflavone are beneficial for yak-bovine interspecies somatic cell nuclear transfer embryo.

    PubMed

    Xiong, X; Li, J; Wang, L; Zhong, J; Zi, X; Wang, Y

    2014-02-01

    With an aim to improve the efficiency of yak-bovine interspecies somatic cell nuclear transfer (iSCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak-bovine iSCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (mSOF) or relative defined culture medium (RDCM) with 5% or 20% oxygen tension. Relative mRNA abundance of Oct-4, IFNT, IGF-2, Bax, GPX-1, SOD-1, CAT and GSS was analysed in blastocysts with qRT-PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension (P < 0.05). The total cell number of blastocyst derived from RDCM with 20% oxygen tension was lower than that of other groups, whereas the group of RDCM with 5% oxygen tension showed a beneficial effect on apoptosis index and tolerance to cryopreservation (P < 0.05). However, under the same oxygen tension, the mRNA abundance of IFNT of RDCM groups was higher than that of the mSOF groups. In addition, high oxygen tension during in vitro culture (IVC) with RDCM significantly increases the mRNA expression of oxidative stress-related genes (GPX-1, SOD-1, CAT and GSS) (P < 0.05). 3, 4-Dihydroxyflavone (DHF) during high oxygen tension was able to improve the cloned blastocyst formation rate in RDCM (P < 0.05). These results for the first time showed that low oxygen tension and RDCM could improve the developmental competence and quality and alleviate the oxidative stress for yak-bovine iSCNT embryo during IVC. © 2013 Blackwell Verlag GmbH.

  18. [Adrenal tumours in childhood].

    PubMed

    Martos-Moreno, G A; Pozo-Román, J; Argente, J

    2013-09-01

    This special article aims to summarise the current knowledge regarding the two groups of tumours with their origin in the adrenal gland: 1) adrenocortical tumours, derived from the cortex of the adrenal gland and 2) phaeochromocytomas and paragangliomas, neuroendocrine tumours derived from nodes of neural crest derived cells symmetrically distributed at both sides of the entire spine (paragangliomas [PG]). These PGs can be functioning tumors that secrete catecholamines, which confers their typical dark colour after staining with chromium salts (chromaffin tumors). Among these, the term phaeochromocytoma (PC) is restricted to those PGs derived from the chromaffin cells in the adrenal medulla (intra-adrenal PGs), whereas the term PG is used for those sympathetic or parasympathetic ones in an extra-adrenal location. We analyse the state of the art of their pathogenic and genetic bases, as well as their clinical signs and symptoms, the tests currently available for performing their diagnosis (biochemical, hormonal, imaging and molecular studies) and management (surgery, pre- and post-surgical medical treatment), considering the current and developing strategies in chemo- and radiotherapy.

  19. Flows of liquid and electrical current through monolayers of cultured bovine arterial endothelium.

    PubMed Central

    Turner, M R

    1992-01-01

    1. Monolayers of arterial endothelium on porous membranes were exposed to a constant pressure between 15 and 35 cmH2O. The rates of liquid flow per unit area (Jv/A) through the monolayers were monitored, together with the electrical resistance (Rm) of the endothelium. 2. At constant pressure, Jv/A decreased with an approximately exponential time course, towards a stable baseline value. This behaviour resembles the sealing previously described for cultured vascular endothelium. At 30-35 cmH2O and 37 degrees C, the mean (+/- S.E.M.) half-time (t1/2) of the decrease in Jv/A (the sealing t1/2) was 548 +/- 141 S (n = 5). The difference between the initial and baseline values of Jv/A was expressed as a fraction of the initial value. The mean (+/- S.E.M.) of this sealing fraction was 0.64 +/- 0.03 (n = 5). Mean (+/- S.E.M.) hydraulic permeability (Lp) was 23.9 +/- 6.4 x 10(-7) cm S-1 cmH2O-1 (n = 9), when measured after sealing. Endothelium appeared damaged after sealing at 30-35 cmH2O and 37 degrees C. 3. Sealing was also observed using glutaraldehyde-fixed endothelium at 30-33 cmH2O and 26-28 degrees C. There was no significant difference between the mean sealing t1/2 of these fixed monolayers, and that of unfixed endothelium at 30-35 cmH2O and 37 degrees C. However, mean sealing fraction was significantly larger for the fixed monolayers than for unfixed endothelium at 30-35 cmH2O and 37 degrees C. There were no significant difference between the post-sealing Lps of these fixed and unfixed monolayers, although the fixed monolayers appeared undamaged after sealing. 4. For unfixed endothelium, Rm was lower after sealing at 30-35 cmH2O and 37 degrees C than before pressure application. There was no significant difference between endothelial Rm before and after sealing, for glutaraldehyde-fixed monolayers. 5. Sealing was also observed at 0 degree C, using unfixed endothelium at 30 cmH2O. Mean sealing t1/2 was not significantly different from that of unfixed endothelium at

  20. What Is Adrenal Cortical Cancer?

    MedlinePlus

    ... include pheochromocytomas (which are most often benign) and neuroblastomas . This document is about tumors and cancers of ... does not discuss tumors of the adrenal medulla. Neuroblastoma s are covered in a separate document . Adrenal cortex ...

  1. Adrenal gland hormone secretion (image)

    MedlinePlus

    The adrenal gland secretes steroid hormones such as cortisol and aldosterone. It also makes precursors that can be converted to ... steroids (androgen, estrogen). A different part of the adrenal gland makes adrenaline (epinephrine). When the glands produce more ...

  2. Congenital adrenal hyperplasia.

    PubMed

    Merke, Deborah P; Bornstein, Stefan R

    Congenital adrenal hyperplasia (CAH) due to deficiency of 21-hydroxylase is a disorder of the adrenal cortex characterised by cortisol deficiency, with or without aldosterone deficiency, and androgen excess. Patients with the most severe form also have abnormalities of the adrenal medulla and epinephrine deficiency. The severe classic form occurs in one in 15,000 births worldwide, and the mild non-classic form is a common cause of hyperandrogenism. Neonatal screening for CAH and gene-specific prenatal diagnosis are now possible. Standard hormone replacement fails to achieve normal growth and development for many children with CAH, and adults can experience iatrogenic Cushing's syndrome, hyperandrogenism, infertility, or the development of the metabolic syndrome. This Seminar reviews the epidemiology, genetics, pathophysiology, diagnosis, and management of CAH, and provides an overview of clinical challenges and future therapies.

  3. The rat adrenal medulla.

    PubMed

    Tischler, A S

    1989-01-01

    Adult adrenal medullary cells, in many strains of rats, develop diffuse and nodular hyperplasia and neoplasia under a variety of conditions. Both endogenous and exogenous factors affect the development of these proliferative changes. The former include the animals' strain, age, and sex. The latter include drugs and other environmental agents, diet, and perhaps stress. Adrenal medullary neoplasms which arise under diverse circumstances often closely resemble each other both morphologically and functionally, and exhibit characteristics of immature chromaffin cells. Recent data indicate that normal, mature-appearing epinephrine- and norepinephrine-type chromaffin cells are able to divide, and suggest that signals which regulate chromaffin cell function also regulate cell proliferation. Prolongation of these signals or superimposed abnormalities might initiate pathological proliferative states. It remains to be determined whether the mechanisms which promote or prevent cell proliferation in the adult adrenal are related to those involved in normal development.

  4. Bone-forming capacity of mesenchymal stromal cells when cultured in the presence of human platelet lysate as substitute for fetal bovine serum.

    PubMed

    Prins, Henk-Jan; Rozemuller, Henk; Vonk-Griffioen, Simone; Verweij, Vivienne G M; Dhert, Wouter J A; Slaper-Cortenbach, Ineke C M; Martens, Anton C M

    2009-12-01

    In tissue engineering, strategies are being developed to repair large bone defects by combining biomaterials and bone marrow-derived multipotent mesenchymal stromal cells (MSCs). For expansion of MSCs under good manufacturing practice conditions, human platelet lysate (PL) can serve as substitute for fetal bovine serum (FBS) in culture media. We compared the in vivo bone-forming capacity of passage 3 MSCs cultured with either PL or FBS for nine different human donors. We also tested the growth kinetics, antigen expression profile, and the multilineage differentiation capacity in vitro of these MSCs. The in vivo bone-forming capacity was determined by seeding culture-expanded MSCs onto biphasic calcium phosphate scaffolds. Hybrid constructs were implanted subcutaneously in nude mice, retrieved after 6 weeks, and analyzed using histomorphometry. PL-supplemented cultures resulted in significantly larger colonies, shorter culture time period, and higher population doublings between P1 and P3 compared to FBS-containing cultures. No differences were observed in antigen expression profiles or differentiation capacities into the osteoblastic, chondrogenic, and adipogenic lineages, qualitatively. In vivo bone formation with PL-supplemented cultures of MSCs was demonstrated in 9/9 donors versus 6/9 for FBS-supplemented cultures. These results warrant the use of PL for ex vivo expansion of human MSCs for bone tissue engineering applications.

  5. Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.

    PubMed

    Christopher-Hennings, Jane; Dammen, Matthew A; Weeks, Shelleen R; Epperson, William B; Singh, Shri N; Steinlicht, Gina L; Fang, Ying; Skaare, Jessica L; Larsen, Jill L; Payeur, Janet B; Nelson, Eric A

    2003-03-01

    In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

  6. Adrenal Gland Disorders: Condition Information

    MedlinePlus

    ... source of sex steroids, such as estrogen and testosterone. What are adrenal gland disorders? Adrenal gland disorders occur when the adrenal glands do not work properly. They can be classified into disorders that occur when too much hormone is produced or when too little hormone is produced. These ...

  7. Adrenal venous sampling in a patient with adrenal Cushing syndrome

    PubMed Central

    Villa-Franco, Carlos Andrés; Román-Gonzalez, Alejandro; Velez-Hoyos, Alejandro; Echeverri-Isaza, Santiago

    2015-01-01

    The primary bilateral macronodular adrenal hyperplasia or the independent adrenocorticotropic hormone bilateral nodular adrenal hyperplasia is a rare cause hypercortisolism, its diagnosis is challenging and there is no clear way to decide the best therapeutic approach. Adrenal venous sampling is commonly used to distinguish the source of hormonal production in patients with primary hyperaldosteronism. It could be a useful tool in this context because it might provide information to guide the treatment. We report the case of a patient with ACTH independent Cushing syndrome in whom the use of adrenal venous sampling with some modifications radically modified the treatment and allowed the diagnosis of a macronodular adrenal hyperplasia. PMID:26309345

  8. Adrenal venous sampling in a patient with adrenal Cushing syndrome.

    PubMed

    Builes-Montaño, Carlos Esteban; Villa-Franco, Carlos Andrés; Román-Gonzalez, Alejandro; Velez-Hoyos, Alejandro; Echeverri-Isaza, Santiago

    2015-01-01

    The primary bilateral macronodular adrenal hyperplasia or the independent adrenocorticotropic hormone bilateral nodular adrenal hyperplasia is a rare cause hypercortisolism, its diagnosis is challenging and there is no clear way to decide the best therapeutic approach. Adrenal venous sampling is commonly used to distinguish the source of hormonal production in patients with primary hyperaldosteronism. It could be a useful tool in this context because it might provide information to guide the treatment. We report the case of a patient with ACTH independent Cushing syndrome in whom the use of adrenal venous sampling with some modifications radically modified the treatment and allowed the diagnosis of a macronodular adrenal hyperplasia.

  9. Effect of leptin supplementation during in vitro oocyte maturation and embryo culture on bovine embryo development and gene expression patterns.

    PubMed

    Arias-Alvarez, M; Bermejo-Alvarez, P; Gutierrez-Adan, A; Rizos, D; Lorenzo, P L; Lonergan, P

    2011-03-15

    Leptin is a metabolic hormone related to body condition and nutritional status that influences fertility in assisted reproductive technologies modulating oocyte and embryo quality. The aim of the present study was to establish the effect of various leptin concentrations (0, 10, 100 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on bovine embryo development and quality in terms of gene expression. The relative mRNA abundance of the genes encoding solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1), Bcl-2-associated X protein (BAX), placenta-specific 8 (PLAC8), aldo-keto reductase family 1 member B1 (AKR1B1) and leptin receptor (LEPR) were determined on Day 7 blastocysts by qRT-PCR. Cleavage rate (P < 0.005) and blastocyst yield (P = 0.05) was significantly lower when cumulus-oocyte complexes (COCs) were matured with 100 ng/mL leptin compared to 0 or 10 ng/mL leptin. No significant effect of different concentrations of leptin added during IVC on blastocyst yield was observed. The presence of 100 ng/mL leptin in both IVM and IVC further decreased cleavage rate (P < 0.005) and blastocyst yield compared to the control group without leptin (P = 0.05) and those supplemented with 10 ng/mL leptin or FCS (P < 0.005). There was no evidence of any leptin-induced difference in the relative transcript abundance of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts. Expression of AKR1B1 was significantly lower in blastocysts from COCs matured with 100 ng/mL leptin compared to those matured with 0 or 10 ng/mL leptin (P < 0.005). LEPR expression was up regulated when leptin concentration was increased from 0 ng/mL during IVM to 10 ng/mL during IVC, but it was down-regulated in the opposite situation (P < 0.005). In conclusion, high leptin concentrations possibly related to obesity seem to be more detrimental rather than the absence of this hormone for preimplantation embryo survival; this effect is independent of LEPR gene

  10. Congenital adrenal hyperplasia

    MedlinePlus

    ... or inappropriately). Congenital adrenal hyperplasia can affect both boys and girls. About 1 in 10,000 to 18,000 ... penis but normal testes Well-developed muscles Both boys and girls will be tall as children, but much shorter ...

  11. Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

    PubMed

    Russell, K A; Koch, T G

    2016-03-01

    Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function. © 2015 EVJ Ltd.

  12. Stimulation of catecholamine synthesis through unique estrogen receptors in the bovine adrenomedullary plasma membrane by 17{beta}-estradiol

    SciTech Connect

    Yanagihara, Nobuyuki . E-mail: yanagin@med.uoeh-u.ac.jp; Liu, Minhui; Toyohira, Yumiko; Tsutsui, Masato; Ueno, Susumu; Shinohara, Yuko; Takahashi, Kojiro; Tanaka, Kazumi

    2006-01-13

    Incubation of cultured bovine adrenal medullary cells with 17{beta}-estradiol (E{sub 2}) (0.3-100 nM) or membrane-impermeable E{sub 2}-bovine serum albumin (100 nM) acutely increased {sup 14}C-catecholamine synthesis from [{sup 14}C]tyrosine. The stimulatory effect of E{sub 2} was not inhibited by ICI182,780, a nuclear estrogen receptor inhibitor. E{sub 2} also increased tyrosine hydroxylase activity and p44/42MAPK phosphorylation, the former of which was attenuated by U0126, an inhibitor of p44/42MAPK kinase. The plasma membrane isolated from the gland showed two classes of specific binding sites of [{sup 3}H]E{sub 2} with apparent K {sub d}s of 3.2 and 106 nM, and B {sub max}s of 0.44 and 8.5 pmol/mg protein, respectively. The high-affinity binding of [{sup 3}H]E{sub 2} was most strongly inhibited by E{sub 2} and phytoestrogens, and to lesser extents by other steroid hormones, while it was enhanced by ICI182,780 and environmental estrogenic pollutants. These findings suggest that E{sub 2} acutely stimulates catecholamine synthesis via activation of p44/42MAPK through unique estrogen receptors in the plasma membrane of bovine adrenal medulla.

  13. Adrenal cortical and medullary imaging.

    PubMed

    Freitas, J E

    1995-07-01

    Adrenal disease can be manifested by endocrine dysfunction or anatomic abnormalities detected by cross-sectional imaging modalities. With the advent of newer and more reliable in vitro assays and a better understanding of the spectrum of adrenal pathology, the physician can now adopt a more accurate and cost-effective approach to the diagnosis of adrenal disease. Both functional and anatomic imaging modalities can play an important role in the evaluation of the incidental adrenal mass, the early detection of adrenal metastases, differentiation of the various causes of Cushings's syndrome, selection of patients for potentially curative surgery in primary aldosteronism and adrenal hyperandrogenism, and localization of pheochromocytomas and neuroblastomas. The usefulness of the adrenal cortical radiopharmaceutical, 131I-6-beta-iodomethylnorcholesterol (NP-59), and the adrenal medullary radiopharmaceuticals, 131I and 123I-metaiodobenzylguanidine (MIBG), is detailed for these various clinical settings and the role of NP-59 and MIBG is contrasted to that of the cross-sectional modalities, computed tomography and magnetic resonance imaging (MRI). Incidental adrenal masses are common, but malignancies are few. Imaging studies select those patients who require a further evaluation by biopsy examination or adrenalectomy. In the hyperfunctioning endocrine states, such as Cushing's syndrome, primary aldosteronism, adrenal androgenism, and pheochromocytoma, correlation of biochemical findings with both functional and anatomic imaging is necessary to avoid inappropriate and ineffective surgical intervention, yet not miss an opportunity for curative resection. Lastly, MIBG and MRI are complementary in the detection and staging of neuroblastoma.

  14. Quantification of Changes in Morphology, Mechanotransduction, and Gene Expression in Bovine Articular Chondrocytes in Response to 2-Dimensional Culture Indicates the Existence of a Novel Phenotype

    PubMed Central

    Qusous, Ala

    2012-01-01

    Objective: Matrix-induced autologous chondrocyte implantation (ACI) offers a potential solution for cartilage repair but is currently hindered by loss of the chondrocyte differentiated phenotype. To further our understanding of the mechanism of dedifferentiation, changes in the phenotype in relation to mechanotransduction were recorded in response to monolayer culture. Methods: Bovine cartilage explants were excised and chondrocytes cultured for 9 days (P1), 14 days (P2), and 21 (P3) days. Changes in morphology and regulatory volume increase (RVI; a mechanotransduction response) were determined by the expression of key genes by RT-PCR and confocal microscopy, respectively. Results: A loss of a differentiated phenotype was observed in P1 with a reduction in sphericity and an overall increase in cell volume from 474.7 ± 32.1 µm3 to 725.2 ± 35.6 µm3. Furthermore, the effect of 2-dimensional (2-D) culture-induced dedifferentiation on mechanotransduction was investigated, whereby RVI and Gd3+-sensitive REV5901-induced calcium rise were only observed in 2-D cultured chondrocytes. A significant up-regulation of types I and II collagens and Sox9 was observed in P1 chondrocytes and no further significant change in type I collagen but a return to baseline levels of type II collagen and Sox9 upon further culture. Conclusion: These data indicated the presence of an intermediate, mesodifferentiated phenotype and highlight the importance of mechanotransduction as a marker of the chondrocytic cell type. PMID:26069635

  15. Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media.

    PubMed

    Souto, Luís I M; Minagawa, Clarice Y; Telles, Evelise O; Garbuglio, Márcio A; Amaku, Marcos; Melville, Priscilla A; Dias, Ricardo A; Sakata, Sonia T; Benites, Nilson R

    2010-02-01

    Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of one or more healthy animals. Mastitis is an inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk in four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman's correlation coefficient was calculated in order to compare the occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacterial-growth mastitis rates and log10 of KF Streptoccocus Agar plate count and there were two positive correlations between coagulase-positive staphylococci and log10 of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

  16. Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos.

    PubMed

    La Rosa, Isabel; Camargo, Luiz S A; Pereira, Michele M; Fernandez-Martin, Rafael; Paz, Dante A; Salamone, Daniel F

    2011-02-01

    BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA) or in vitro fertilization (IVF) and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100 ng/ml of BMP4 or Noggin in CR2 until day nine of culture. Cleavage, blastocyst and hatching rates, expression pattern of the transcription factor Oct-4 in blastocysts and embryo cell number at day two and nine post-activation or fertilization were evaluated. We found that Noggin, as BMP4, did not affect oocyte nuclear maturation. Noggin supplementation up-regulated the expression of HSP70 and MATER genes in matured oocytes. Moreover, BMP4 during maturation increased the proportion of Oct-4 positive cells in parthenogenic embryos. On the other hand, when Noggin was added to embryo culture medium, developmental rates of parthenogenic and in vitro fertilized embryos were reduced. However, BMP4 addition decreases the development only for in vitro fertilized embryos. BMP4 and Noggin during culture reduced the proportion of Oct-4-expressing cells. Our results show that BMP4 is implicated in bovine oocytes maturation and embryo development. Moreover, our findings demonstrate, for the first time, that a correct

  17. CT demonstration of bilateral adrenal hemorrhage

    SciTech Connect

    Ling, D.; Korobkin, M.; Silverman, P.M.; Dunnick, N.R.

    1983-08-01

    Bilateral adrenal hemorrhage with subsequent adrenal insufficiency is a recognized complication of anticoagulant therapy. Because the clinical manifestations are often nonspecific, the antemortem diagnosis of adrenal hemorrhage has been a difficult clinical problem. Computed tomography (CT) provides detailed images of the adrenal glands that are not possible with conventional imaging methods. The CT findings of bilateral adrenal hemorrhage in an anticoagulated patient are reported.

  18. Equine bone marrow mesenchymal or amniotic epithelial stem cells as feeder in a model for the in vitro culture of bovine embryos.

    PubMed

    Lange-Consiglio, Anna; Maggio, Valentina; Pellegrino, Laura; Cremonesi, Fausto

    2012-02-01

    Various studies have shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In the present study we investigated the effects of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSCs) or equine amniotic epithelial stem cells (AE-SCs) on in vitro blastocysts development. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and the isolated cells were resuspended in Dulbecco Modified Earle's Medium supplemented with 10 ng/ml of basic fibroblast growth factor (bFGF). Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fragments were incubated with 0.05% trypsin for 45 min. Separated AE cells were plated in standard culture medium containing 10 ng/ml epidermal growth factor (EGF). Seven hundred and five cumulus-oocyte complexes were used and, after IVM and IVF, cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured up to day 7: (1) co-culture with cumulus cells (control); (2) co-culture with BM-MSCs; and (3) co-culture with AE-SCs. Statistical analyses were performed by ANOVA. Blastocyst developmental rates were significantly different (p < 0.001) between control, AE-SCs and BM-MSCs (respectively 35.45, 41.84 and 30.09%). In conclusion, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSCs and cumulus cells. It can be suggested that these cells secrete biologically active substances, including signalling molecules and growth factors of epithelial nature, different to those of the BM cells of mesenchymal origin.

  19. Set up of a serum-free culture system for bovine embryos: embryo development and quality before and after transient transfer.

    PubMed

    George, F; Daniaux, C; Genicot, G; Verhaeghe, B; Lambert, P; Donnay, I

    2008-03-15

    It is well known that serum in culture medium negatively affects blastocyst quality. The objective of this work was to develop and test a serum-free culture medium which could improve embryo quality, measured by the resistance to freezing, lipid and glutathione content of the resulting blastocysts, as well as the ability of the blastocysts to elongate after transient transfer to recipient cows. In a first experiment we showed that adding a mixture of insulin, transferrin and selenium to serum-free Synthetic Oviduct Fluid medium (SOF-ITS) improved embryo development and quality. In the second experiment, the addition of BSA to SOF-ITS further improved blastocyst development. Moreover, a reduction in lipid content of morulae was observed in SOF-ITS-BSA by comparison with morulae cultured with serum (SOF-FCS). The resistance to freezing measured by hatching rates 24h post-thawing was also improved for blastocysts with a diameter between 160 and 180 microm cultured in SOF-ITS-BSA by comparison to those produced with serum. In order to evaluate the redox potential of the embryos, reduced glutathione content (GSH) was evaluated both before and after cryopreservation. A significant decrease in glutathione was observed after freezing, whatever the culture medium, but no difference was observed between culture conditions. Transient transfers were performed and elongated D-13 embryos were recovered. Elongation was more pronounced and the embryonic disk more often visible in embryos cultured in SOF-ITS-BSA than in embryos cultured with FCS. In conclusion, the serum-free system we developed to produce in vitro bovine embryos meets the developmental and qualitative requirements for a large-scale use.

  20. Congenital lipoid adrenal hyperplasia

    PubMed Central

    2014-01-01

    Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most fatal form of CAH, as it disrupts adrenal and gonadal steroidogenesis. Most cases of lipoid CAH are caused by recessive mutations in the gene encoding steroidogenic acute regulatory protein (StAR). Affected patients typically present with signs of severe adrenal failure in early infancy and 46,XY genetic males are phenotypic females due to disrupted testicular androgen secretion. The StAR p.Q258X mutation accounts for about 70% of affected alleles in most patients of Japanese and Korean ancestry. However, it is more prevalent (92.3%) in the Korean population. Recently, some patients have been showed that they had late and mild clinical findings. These cases and studies constitute a new entity of 'nonclassic lipoid CAH'. The cholesterol side-chain cleavage enzyme, P450scc (CYP11A1), plays an essential role converting cholesterol to pregnenolone. Although progesterone production from the fetally derived placenta is necessary to maintain a pregnancy to term, some patients with P450scc mutations have recently been reported. P450scc mutations can also cause lipoid CAH and establish a recently recognized human endocrine disorder. PMID:25654062

  1. Radioguided Adrenal Surgery

    PubMed Central

    Deus, Javier; Millera, Alfonso; Andrés, Alejandro; Prats, Enrique; Gil, Ismael; Suarez, Manuel; Salcini, José L.; Lahoz, Manuel

    2015-01-01

    Abstract The laparoscopic adrenalectomy is considered as the procedure of choice for the treatment of adrenal hyperplasia and tumor lesions. However, some special situations may limit the use of this method due to the difficulty to locate the gland and perform the lesion excision. We analyze 2 patients of a left adrenal tumor, explaining how they have overcome the difficulties in both situations. The first case was a patient with a history of intra-abdominal surgery and the other patient suffered from severe obesity. We performed with the use of the gamma probe, and the 2 cases, was of great help to access and glandular localization. The help of gamma probe test was achieved in the surgical bed, that removal was complete. The use of the portable gamma probe facilitated the access to the left adrenal gland as well as conducting the glandular excision without delay, despite the difficulties due to the intra abdominal surgery caused by the previous surgery, and in the case of severe obesity. PMID:26426608

  2. Transmission ratio distortion at the growth hormone gene (GH1) in bovine preimplantation embryos: An in vitro culture-induced phenomenon?

    PubMed

    Murphy, Angela M; Meade, Kieran G; Hayes, Patricia A; Park, Stephen D E; Evans, Alex C O; Lonergan, Patrick; MacHugh, David E

    2008-05-01

    The growth hormone gene (GH1) and its polypeptide product (GH) have a crucial role in reproduction, embryogenesis and general development. A polymorphism present in the fifth exon of the bovine GH1 gene (GH1 p.Leu127Val) has been associated with GH release and milk production in cattle. The objective of the present study was to examine the genotype frequencies of the GH1 p.Leu127Val polymorphism in bovine blastocysts produced in vitro and in vivo to determine if allelic variation of the GH1 gene affects embryo development and survival. A heterozygous (p.Leu127/Val127) sire was used for in vitro fertilization of oocytes of unknown maternal genotype (n = 104) and known maternal genotype (n = 115). PCR amplification and genotyping of the GH1 gene from Day 8 blastocysts derived from these fertilized oocytes demonstrated that there was significant over-representation from the expected Mendelian ratio of GH1 p.Leu127/Leu127 homozygotes from oocytes of known maternal genotype (P = 0.006). Contrary to this, analysis of in vivo-produced bovine blastocysts of known parental GH1 genotype (n = 69) did not reveal an overrepresentation of GH1 p.Leu127/Leu127 homozygotes. These results suggest that developing in vitro-produced embryos are exposed to a selection process, probably due to a less favorable culture environment, that acts to increase the number of GH1 p.Leu127/Leu127 homozygotes, thereby giving rise to the observed transmission ratio distortion (TRD) of GH1 genotypes when compared to in vivo produced embryos.

  3. Development of adrenal cortex zonation.

    PubMed

    Xing, Yewei; Lerario, Antonio M; Rainey, William; Hammer, Gary D

    2015-06-01

    The human adult adrenal cortex is composed of the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), which are responsible for production of mineralocorticoids, glucocorticoids, and adrenal androgens, respectively. The final completion of cortical zonation in humans does not occur until puberty with the establishment of the zR and its production of adrenal androgens; a process called adrenarche. The maintenance of the adrenal cortex involves the centripetal displacement and differentiation of peripheral Sonic hedgehog-positive progenitors cells into zG cells that later transition to zF cells and subsequently zR cells.

  4. [Sonography of the adrenal glands].

    PubMed

    Rüeger, R

    2005-03-02

    In the abdominal ultrasonography, the representation of normal adrenal glands is frequently problematic, also for experienced practitioners in ultrasonography. During a seminary at the congress of the SGUM in Davos, in June 2004, it was specially entered to this problematic by anatomical illustrations and live demonstrations. These statements will be summarized in the following article. Also, the technics of examination of the adrenal glands will be explained, especially in comparison to anatomical cut-preparations. It will be entered to particular pathological statements of the adrenal glands. The proceeding will be described by the localisation of accidentally detected tumours of adrenal glands.

  5. Adrenal crisis secondary to bilateral adrenal haemorrhage after hemicolectomy

    PubMed Central

    Tsang, Venessa H M; Kabir, Shahrir; Ip, Julian C Y

    2016-01-01

    Summary Adrenal haemorrhage is a rare cause of adrenal crisis, which requires rapid diagnosis, prompt initiation of parenteral hydrocortisone and haemodynamic monitoring to avoid hypotensive crises. We herein describe a case of bilateral adrenal haemorrhage after hemicolectomy in a 93-year-old female with high-grade colonic adenocarcinoma. This patient’s post-operative recovery was complicated by an acute hypotensive episode, hypoglycaemia and syncope, and subsequent computed tomography (CT) scan of the abdomen revealed bilateral adrenal haemorrhage. Given her labile blood pressure, intravenous hydrocortisone was commenced with rapid improvement of blood pressure, which had incompletely responded with fluids. A provisional diagnosis of hypocortisolism was made. Initial heparin-induced thrombocytopenic screen (HITTS) was positive, but platelet count and coagulation profile were both normal. The patient suffered a concurrent transient ischaemic attack with no neurological deficits. She was discharged on a reducing dose of oral steroids with normal serum cortisol levels at the time of discharge. She and her family were educated about lifelong steroids and the use of parenteral steroids should a hypoadrenal crisis eventuate. Learning points: Adrenal haemorrhage is a rare cause of hypoadrenalism, and thus requires prompt diagnosis and management to prevent death from primary adrenocortical insufficiency. Mechanisms of adrenal haemorrhage include reduced adrenal vascular bed capillary resistance, adrenal vein thrombosis, catecholamine-related increased adrenal blood flow and adrenal vein spasm. Standard diagnostic assessment is a non-contrast CT abdomen. Intravenous hydrocortisone and intravenous substitution of fluids are the initial management. A formal diagnosis of primary adrenal insufficiency should never delay treatment, but should be made afterwards. PMID:27855238

  6. Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

    PubMed

    Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

    2010-12-01

    We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

  7. Ex Vivo bone formation in bovine trabecular bone cultured in a dynamic 3D bioreactor is enhanced by compressive mechanical strain.

    PubMed

    David, Valentin; Guignandon, Alain; Martin, Aline; Malaval, Luc; Lafage-Proust, Marie-Hélène; Rattner, Aline; Mann, Val; Noble, Brendon; Jones, David B; Vico, Laurence

    2008-01-01

    Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Young's modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.

  8. Chinese hamster ovary cells cultured in low concentrations of fetal bovine serum: cloning efficiency, growth in suspension, and selection of drug-resistant mutant phenotypes

    SciTech Connect

    Carver, J.H.; Salazar, E.P.; Knize, M.G.

    1983-09-01

    Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used for applied mutagenesis assays, cloning efficiency and growth parameters in suspension of Chinese hamster ovary cells cultured were measured in reduced serum with or without additives (1 ..mu..g/ml insulin, 3 x 10/sup -7/ M linoleic acid, 1 x 10/sup -8/ M H/sub 2/SeO/sub 3/) or bovine serum albumin (BSA, 1% wt/vol). With the additives and less than or equal to 0.5% fetal bovine serum (FBS), Ham's F12 medium (without hypoxanthine and thymidine) was more optimal than alpha Eagle's minimum essential medium (MEM) (without ribosides and deoxyribosides) for low density cloning and high density suspension growth. The spontaneous frequency of azaadenine-resistant phenotypes (mutant at the aprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxy-uridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM with 2% FBS plus BSA; observed mutant frequencies induced by dimethylnitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.

  9. Addition of erythrocytes to in vitro culture medium attenuates the detrimental effects of reactive oxygen species on bovine preimplantation embryo development.

    PubMed

    Ideta, Atsushi; Tsuchiya, Kanami; Aoyagi, Yoshito

    2012-01-01

    Erythrocytes were recently found to improve the early development of mice embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine in vitro fertilized (IVF) embryos in medium supplemented with reactive oxygen species (ROS). IVF embryos were cultured in CR1aa medium supplemented with oxidizing agents, 0.5mmol/L hypoxanthine and 0.01U/mL xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5×10(4) , 5×10(5) , 5×10(6) and 5×10(7) erythrocytes/mL). After 8 days, blastocysts were examined with a stereomicroscope. HX/XOD blocked development to the blastocyst stage (HX/XOD: 0%, control: 33%), but in the presence of both erythrocytes and HX/XOD, blastocyst development was restored to about one-third to two-thirds the normal rate (5×10(5) to 5×10(7) erythrocytes/mL: 12 to 23%). Furthermore, adding erythrocytes or erythrocyte hemolysate to medium without HX/XOD increased the blastocyst rate. These results suggest that the addition of erythrocytes can attenuate the detrimental effects of ROS on embryo development in bovine species as well as in mice.

  10. Traumatic and non-traumatic adrenal emergencies.

    PubMed

    Chernyak, Victoria; Patlas, Michael N; Menias, Christine O; Soto, Jorge A; Kielar, Ania Z; Rozenblit, Alla M; Romano, Luigia; Katz, Douglas S

    2015-12-01

    Multiple traumatic and non-traumatic adrenal emergencies are occasionally encountered during the cross-sectional imaging of emergency department patients. Traumatic adrenal hematomas are markers of severe polytrauma, and can be easily overlooked due to multiple concomitant injuries. Patients with non-traumatic adrenal emergencies usually present to an emergency department with a non-specific clinical picture. The detection and management of adrenal emergencies is based on cross-sectional imaging. Adrenal hemorrhage, adrenal infection, or rupture of adrenal neoplasm require immediate detection to avoid dire consequences. More often however, adrenal emergencies are detected incidentally in patients being investigated for non-specific acute abdominal pain. A high index of suspicion is required for the establishment of timely diagnosis and to avert potentially life-threatening complications. We describe cross-sectional imaging findings in patients with traumatic and non-traumatic adrenal hemorrhage, adrenal infarctions, adrenal infections, and complications of adrenal masses.

  11. Adrenal glands transabdominal ultrasonography - pictorial essay.

    PubMed

    Chira, Romeo Ioan; Chira, Alexandra; Manzat-Saplacan, Roberta Maria; Nagy, Georgiana; Valea, Ana; Silaghi, Alina Cristina; Mircea, Petru Adrian; Valean, Simona

    2017-05-03

    Adrenal gland ultrasonography is one of the corner stones of the abdominal ultrasonography examination for many medical specialties. The adrenal areas can be easily overlooked though adrenal gland pathology is diverse. We present the normal aspects and various transabdominal ultrasonography findings of the adrenal glands, both common and rare. Even though ultrasound examination is operator and patient dependent, we consider the examination of the adrenal glands very important, due to relatively frequent incidental detection of an adrenal mass.

  12. Extracellular Ionic Composition Alters Kinetics of Vesicular Release of Catecholamines and Quantal Size During Exocytosis at Adrenal Medullary Cells

    DTIC Science & Technology

    1994-07-05

    cytosolic calcium concentration in single bovine adrenal chromaffin cells form video imaging of fura - 2 , EMBO J. 8, 401-411. Pollard, H. B., Ornberg, R...Key words: chromaffin cells, catecholamine, barium induced exocytosis, fura - 2 , calcium independent exocytosis Running title: Effects of extracellular...calibration of the fluorescent calcium indicator Fura - 2 , Cell Calcium 11, 75-83. Winkler, H. (1976) The Composition of Adrenal Chromaffin Granules

  13. Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue.

    PubMed

    Sorg, D; Potzel, A; Beck, M; Meyer, H H D; Viturro, E; Kliem, H

    2012-10-01

    Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

  14. Evaluation of different culture systems with low oxygen tension on the development, quality and oxidative stress-related genes of bovine embryos produced in vitro.

    PubMed

    Arias, Maria Elena; Sanchez, Raul; Felmer, Ricardo

    2012-08-01

    The present study was conducted to assess the development, quality and gene expression profile of oxidative stress-related genes of bovine embryos cultured in different culture systems with low oxygen tension (5% CO2, 5% O2 and 90% N2). The systems assessed included: (1) an incubator chamber; (2) a plastic bag; and (3) a foil bag. The choice of culture system had no effect on cleavage rate at 72 h. However, significant differences (P < 0.01) were observed in the rate of blastocysts registered at day 7 (29.8, 20.2 and 12.7% for incubator chamber, plastic bag and foil bag, respectively). Total number of cells did not differ between systems, although the proportion of ICM:total cells was affected particularly in the plastic bag (19.5%), compared with the incubator chamber (31.4%). In addition, significant differences were found in the apoptotic:total cell ratio (3.3, 6.5 and 8.8% for the incubator chamber, plastic bag and foil bag, respectively), with apoptotic nuclei localised mainly in the ICM compartment of the embryo. The amount of reactive oxygen species was also different between culture systems and this effect was correlated with a higher expression of SOD2, GSS and GPX1 genes in embryos cultured in the gassed bags as compared with embryos cultured in the incubator chamber. In conclusion, these results give evidence that, under low oxygen tension, the incubator chamber is more efficient and generates higher number of, and better quality, embryos than gassed bag systems evaluated here and this effect was probably due to an increased level of reactive oxygen species in the gassed bags, which upregulates the expression of some antioxidant enzymes to compensate for hyperoxia conditions.

  15. Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

    PubMed

    Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

    2016-01-01

    Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin

  16. Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model

    PubMed Central

    Komninou, Eliza Rossi; Remião, Mariana Härter; Lucas, Caroline Gomes; Domingues, William Borges; Basso, Andrea Cristina; Jornada, Denise Soledade; Deschamps, João Carlos; Beck, Ruy Carlos Ruver; Pohlmann, Adriana Raffin; Bordignon, Vilceu; Seixas, Fabiana Kömmling; Campos, Vinicius Farias; Guterres, Silvia Stanisçuaski; Collares, Tiago

    2016-01-01

    Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10−6, 10−9, and 10−12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10−9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10−9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of

  17. [Adrenal carcinoma induced hypoglycemia].

    PubMed

    Soutelo, Jimena; Saban, Melina; Borghi Torzillo, Florencia; Lutfi, Ruben; Leal Reyna, Mariela

    2013-01-01

    Adrenal carcinoma is a rare malignancy of poor prognosis. The most common clinical presentation is secondary to hormone production, while the development of symptomatic hypoglycemia is exceptional. We report the case of a 37 year old-woman admitted to hospital with severe hypoglycemia, hypertension, hypokalemia and amenorrhea. In the laboratory we found hypoglycemia, with low insulin levels, and androgen levels in tumor range. CT of abdomen and pelvis showed a heterogeneous lesion of solid appearance without a cleavage plane relative to liver parenchyma, and intense contrast enhancement. Retroperitoneal mass was removed, and the patient evolved without complications, blood glucose and potassium were normalized, blood pressure stabilized and menstrual cycles recovered.

  18. Liver x receptors stimulate lipogenesis in bovine mammary epithelial cell culture but do not appear to be involved in diet‐induced milk fat depression in cows

    PubMed Central

    Harvatine, Kevin J.; Boisclair, Yves R.; Bauman, Dale E.

    2014-01-01

    Abstract Milk fat synthesis of ruminants can be inhibited by intermediates of ruminal fatty acid biohydrogenation including trans‐10, cis‐12 conjugated linoleic acid (CLA). These biohydrogenation intermediates signal a coordinated downregulation of genes involved in mammary FA synthesis, transport, and esterification. We have previously reported decreased mammary expression of sterol response element‐binding protein 1 (SREBP1), SREBP1‐activating proteins, and thyroid hormone‐responsive spot 14 (S14) in the cow during diet‐induced milk fat depression (MFD), and treatment with trans‐10, cis‐12 CLA. Liver x receptors (LXR) and retinoid x receptors (RXR) regulate lipogenesis and are known to bind polyunsaturated FA and LXR agonist increases lipid synthesis in mammary epithelial cell culture. The current studies investigated if biohydrogenation products of rumen origin inhibit mammary lipogenesis through LXR and/or RXR. Expression of LXRs was not different in lactating compared to nonlactating bovine mammary tissue, and expression of LXRs, RXRα, and selected LXR and RXR target genes was not changed in mammary tissue during diet‐induced or CLA‐induced MFD in the cow. In bovine mammary epithelial cell culture, LXR agonist stimulated lipogenesis and expression of LXRß, ATP‐binding cassette 1 (ABCA1), SREBP1c, and S14, but LXR activation did not overcome CLA inhibition of lipogenesis and downregulation of LXRß, SREBP1c, and S14 expression. Lastly, expression of the LXR‐regulated carbohydrate‐responsive element‐binding protein (ChREBP) was higher in lactating than nonlactating tissue and was decreased during CLA‐induced MFD. We conclude that changes in mammary LXR expression in dairy cows are not involved in MFD and that trans‐10, cis‐12 CLA inhibition of lipogenesis and diet‐induced MFD appears independent of direct LXR signaling. PMID:24760520

  19. Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos.

    PubMed

    Merton, J S; Vermeulen, Z L; Otter, T; Mullaart, E; de Ruigh, L; Hasler, J F

    2007-04-15

    Many environmental conditions for in vitro embryo production (IVP) systems for cattle have been relatively standardised, e.g. media composition, temperature, pH, water quality, and atmospheric composition. However, little attention has been paid to the quality of ambient laboratory air and the gas environment in incubators. Although a few studies have examined the effects of chemical air contamination on IVP of human embryos, there are no published accounts for domestic animal embryos. Therefore, this study investigated the effects of an intra-incubator carbon-activated air filtration system (CODA) during in vitro culture (IVC) on embryonic development and subsequent pregnancy rate of bovine embryos. Immature cumulus-oocyte-complexes (COCs) were obtained twice-weekly by ultrasonic-guided transvaginal oocyte aspiration. The COCs were matured in TCM199/FCS/LH/FSH, fertilized with frozen-thawed Percoll-separated semen, and subsequently cultured for 7 day in SOFaaBSA. Day 7 embryos were transferred either fresh or frozen/thawed. The experimental design was a 2 x 2 factorial; presumptive zygotes were placed either in a conventional CO(2)-O(2)-N(2) incubator (Control group) or in an identical CO(2)-O(2)-N(2) incubator with a CODA intra-incubator air purification unit (CODA group) for IVC. The embryo production rate at Day 7 was not affected by the CODA air purification unit (23.4 and 24.7% morulae and blastocysts per oocyte for control and CODA, respectively) nor was there any significant effect on embryo stage or quality. However, the pregnancy rate was improved (P=0.043) for both fresh (46.3% versus 41.0%) and frozen/thawed embryos (40.8% versus 35.6%). In conclusion, atmospheric purification by the CODA intra-incubator air purification unit significantly increased pregnancy rate following transfer of in vitro-produced bovine embryos.

  20. The effects of ACTH on steroid metabolomic profiles in human adrenal cells.

    PubMed

    Xing, Yewei; Edwards, Michael A; Ahlem, Clarence; Kennedy, Mike; Cohen, Anthony; Gomez-Sanchez, Celso E; Rainey, William E

    2011-06-01

    The adrenal glands are the primary source of mineralocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of 23 steroid hormones in adult adrenal primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis revealed that, in primary adrenal cell cultures, cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48  h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11β-hydroxyandrostenedione (11OHA), that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells, 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11β-hydroxylated products, including cortisol and 11OHA. This study illustrates that adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.

  1. Sensitivity and specificity of on-farm scoring systems and nasal culture to detect bovine respiratory disease complex in preweaned dairy calves.

    PubMed

    Love, William J; Lehenbauer, Terry W; Van Eenennaam, Alison L; Drake, Christiana M; Kass, Philip H; Farver, Thomas B; Aly, Sharif S

    2016-03-01

    The California (CA) and Wisconsin (WI) clinical scoring systems have been proposed for bovine respiratory disease complex (BRDC) detection in preweaned dairy calves. The screening sensitivity (SSe), for estimating BRDC prevalence in a cohort of calves, diagnostic sensitivity (DSe), for confirming BRDC in ill calves, and specificity (Sp) were estimated for each of the scoring systems, as well as for nasal swab cultures for aerobic bacteria and mycoplasma species. Thoracic ultrasound and auscultation were used as the reference standard tests interpreted in parallel. A total of 536 calves (221 with BRDC and 315 healthy) were sampled from 5 premises in California. The SSe of 46.8%, DSe of 72.6%, and Sp of 87.4% was determined for the CA system. The SSe of 46.0%, DSe of 71.1%, and Sp of 91.2% was determined for the WI system. For aerobic culture, the SSe was 43.4%, DSe was 52.6%, and Sp was 71.3%; for Mycoplasma spp. culture, the SSe was 57.5%, DSe was 68.9%, and Sp was 59.7%. The screening and diagnostic sensitivities of the scoring systems were not significantly different but the Sp of the WI system was greater by 3.8%. Scoring systems can serve as rapid on-farm tools to determine the burden of BRDC in preweaned dairy calves. However, users may expect the SSe to be less than the DSe when confirming BRDC in an ill calf.

  2. Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture.

    PubMed Central

    Canfield, A E; Schor, A M; West, D C; Schor, S L; Grant, M E

    1987-01-01

    Biosynthetic experiments with cultured bovine retinal endothelial cells have identified a glycoprotein of Mr 47,000 (Gp47) as a major component secreted into the medium. Gp47 is a non-collagenous glycoprotein with a pI of 4.6-5.5, which does not bind to either gelatin-Sepharose or heparin-Sepharose but is retained by concanavalin A-Sepharose. The Mr of this species decreases to approx. 42,000 in the presence of tunicamycin, indicating that it contains asparagine-linked oligosaccharides. A second protein of Mr 47,000 (P47) is present in the cell layer/matrix of these cultured cells. The electrophoretic mobility of P47 remains unaltered when synthesized in the presence of tunicamycin. Peptide-mapping experiments using N-chlorosuccinimide and Staphylococcus aureus V8 proteinase demonstrate that Gp47 and P47 are distinct proteins, and are not related to colligin, a membrane-bound collagen-receptor protein of similar size, or to SPARC, a major secreted product of parietal endodermal cells and sparse cultures of aortic endothelial cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:3675551

  3. Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

    PubMed Central

    Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

    2017-01-01

    Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation. PMID:28465691

  4. The bovine peripheral-type benzodiazepine receptor: A receptor with low affinity for benzodiazepines

    SciTech Connect

    Parola, A.L.; Laird, H.E. II )

    1991-01-01

    The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit ({sup 3}H)PK 11195 binding was PK 11195 > protoporphyrin IX > benzodiazepines. ({sup 3}H)PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. ({sup 3}H)PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing and denaturing conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.

  5. Elevated luteinizing hormone induces expression of its receptor and promotes steroidogenesis in the adrenal cortex

    PubMed Central

    Kero, Jukka; Poutanen, Matti; Zhang, Fu-Ping; Rahman, Nafis; McNicol, Anne Marie; Nilson, John H.; Keri, Ruth A.; Huhtaniemi, Ilpo T.

    2000-01-01

    Transgenic (TG) female mice expressing bLHβ-CTP (a chimeric protein derived from the β-subunit of bovine luteinizing hormone [LH] and a fragment of the β-subunit of human chorionic gonadotropin [hCG]) exhibit elevated serum LH, infertility, polycystic ovaries, and ovarian tumors. In humans, increased LH secretion also occurs in infertility and polycystic ovarian syndrome, often concomitant with adrenocortical dysfunction. We therefore investigated adrenal function in LH overexpressing bLHβ-CTP female mice. The size of their adrenals was increased by 80% with histological signs of cortical stimulation. Furthermore, adrenal steroid production was increased, with up to 14-fold elevated serum corticosterone. Primary adrenal cells from TG and control females responded similarly to ACTH stimulation, but, surprisingly, the TG adrenals responded to hCG with significantly increased cAMP, progesterone, and corticosterone production. LH receptor (LHR) expression and activity were also elevated in adrenals from female TG mice, but gonadectomized TG females showed no increase in corticosterone, suggesting that the dysfunctional ovaries of the intact TG females promote adrenocortical hyperfunction. We suggest that, in intact TG females, enhanced ovarian estrogen synthesis causes increased secretion of prolactin (PRL), which elevates LHR expression. Chronically elevated serum LH, augmented by enhanced PRL production, induces functional LHR expression in mouse adrenal cortex, leading to elevated, LH-dependent, corticosterone production. Thus, besides polycystic ovaries, the bLHβ-CTP mice provide a useful model for studying human disorders related to elevated LH secretion and adrenocortical hyperfunction. PMID:10712435

  6. Elevated luteinizing hormone induces expression of its receptor and promotes steroidogenesis in the adrenal cortex.

    PubMed

    Kero, J; Poutanen, M; Zhang, F P; Rahman, N; McNicol, A M; Nilson, J H; Keri, R A; Huhtaniemi, I T

    2000-03-01

    Transgenic (TG) female mice expressing bLHbeta-CTP (a chimeric protein derived from the beta-subunit of bovine luteinizing hormone [LH] and a fragment of the beta-subunit of human chorionic gonadotropin [hCG]) exhibit elevated serum LH, infertility, polycystic ovaries, and ovarian tumors. In humans, increased LH secretion also occurs in infertility and polycystic ovarian syndrome, often concomitant with adrenocortical dysfunction. We therefore investigated adrenal function in LH overexpressing bLHbeta-CTP female mice. The size of their adrenals was increased by 80% with histological signs of cortical stimulation. Furthermore, adrenal steroid production was increased, with up to 14-fold elevated serum corticosterone. Primary adrenal cells from TG and control females responded similarly to ACTH stimulation, but, surprisingly, the TG adrenals responded to hCG with significantly increased cAMP, progesterone, and corticosterone production. LH receptor (LHR) expression and activity were also elevated in adrenals from female TG mice, but gonadectomized TG females showed no increase in corticosterone, suggesting that the dysfunctional ovaries of the intact TG females promote adrenocortical hyperfunction. We suggest that, in intact TG females, enhanced ovarian estrogen synthesis causes increased secretion of prolactin (PRL), which elevates LHR expression. Chronically elevated serum LH, augmented by enhanced PRL production, induces functional LHR expression in mouse adrenal cortex, leading to elevated, LH-dependent, corticosterone production. Thus, besides polycystic ovaries, the bLHbeta-CTP mice provide a useful model for studying human disorders related to elevated LH secretion and adrenocortical hyperfunction.

  7. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  8. 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.

    PubMed

    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

  9. [Lumbar pain and bilateral adrenal masses].

    PubMed

    García, Elena; Sánchez, Raquel; Martínez, Guillermo; Bernal, Carmen; Calatayud, M; Partida, M; Hawkins, Federico

    2009-05-01

    Many problems may arise when defining whether adrenal lesions are primary to the adrenal glands or represent other tissue, whether they are benign or malignant and whether they are functioning or nonfunctioning. Adrenal imaging complements the clinical and hormonal evaluation of these patients. We present a patient with lumbar pain and bilateral adrenal masses.

  10. Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair.

    PubMed

    Park, Yongdoo; Lutolf, Matthias P; Hubbell, Jeffrey A; Hunziker, Ernst B; Wong, Marcy

    2004-01-01

    A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

  11. Adrenal involvement in non-Hodgkin lymphoma

    SciTech Connect

    Paling, M.R.; Williamson, B.R.J.

    1983-08-01

    Adrenal masses are described in seven cases of non-Hodgkin lymphoma in a series of 173 patients. In all seven patients the lymphoma was diffuse rather than nodular. Three patients had adrenal masses at the time of presentation, whereas in four cases the adrenal gland was a site of tumor recurrence after therapy. Three patients had simultaneous bilateral adrenal involvement by tumor. No characteristic features were recognized that might have distinguished these tumors from other adrenal masses. Appropriate therapy successfully resolved the adrenal masses in all but one case. The latter patient was the only one with evidence of adrenal insufficiency.

  12. Repair of wounded monolayers of cultured bovine aortic endothelial cells is inhibited by calcium spirulan, a novel sulfated polysaccharide isolated from Spirulina platensis.

    PubMed

    Kaji, Toshiyuki; Fujiwara, Yasuyuki; Inomata, Yuki; Hamada, Chieko; Yamamoto, Chika; Shimada, Satomi; Lee, Jung-Bum; Hayashi, Toshimitsu

    2002-03-08

    Calcium spirulan (Ca-SP) is a novel sulfated polysaccharide isolated from a blue-green alga Spirulina platensis. Ca-SP inhibits thrombin by activation of heparin cofactor II. Therefore, it could serve as an origin of anti-atherogenic medicines. Since maintenance of vascular endothelial cell monolayers is important for prevention of vascular lesions such as atherosclerosis, the effect of Ca-SP at 20 microg/ml or less on the repair of wounded bovine aortic endothelial cell monolayers in culture was investigated in the present study. When the monolayers were wounded and cultured in the presence of Ca-SP, the polysaccharide inhibited the appearance of the cells in the wounded area. The inhibition was also observed even when the repair was promoted by excess basic fibroblast growth factor, which is one of the autocrine growth factors that are involved in the endothelial cell monolayer maintenance. On the other hand, Ca-SP inhibited the cell growth and the incorporation of [3H]thymidine into the acid-insoluble fraction of proliferating endothelial cells, suggesting that Ca-SP inhibits endothelial cell proliferation. From these results, it is concluded that Ca-SP may retard the repair process of damaged vascular endothelium through inhibition of vascular endothelial cell proliferation by induction of a lower ability to respond to stimulation by endogenous basic fibroblast growth factor.

  13. Laparoendoscopic single site adrenal surgery.

    PubMed

    Han, Deok Hyun; Lim, Meng Shi; Seo, Joo Wan; Jeong, Byong Chang; Rha, Koong Ho

    2012-04-01

    Laparoscopic adrenal surgery is a standard procedure for the management of benign adrenal pathology and small malignant tumors. There has been an increasing interest over the last few years in the use of laparoendoscopic single-site surgery (LESS). From recent literatures, LESS adrenalectomy was demonstrated that this technique was safe and feasible despite the relatively difficult anatomical location of the adrenal gland. We reviewed the surgical techniques and outcomes of LESS adrenalectomy including robot-assisted approach and gave an overview of the current role of LESS in adrenalectomy.

  14. [Hormonal evaluation of adrenal incidentalomas].

    PubMed

    Le Loupp, Anne-Gaëlle; Cariou, Bertrand; Drui, Delphine; Graveline, Nolwenn; Masson, Damien; Bach-Ngohou, Kalyane

    2014-01-01

    Imaging technological advances and widespread use of abdominal imaging have led to the identification of an increasing number of adrenal incidentalomas in the last decades. Causes of these adrenal masses are multiple, but the most common etiology is the non-functional adenoma. Although in most cases, these masses are benign and non-functional, clinicians have to perform biochemical testing for subclinical cushing's syndrome, pheochromocytoma or primary hyperaldosteronism. This screening is essential for their etiological diagnosis and therapeutic management. We report in this article the biological approach to detect secretory activity of adrenal incidentalomas and the diagnostic accuracy of the biochemical tests used.

  15. Stimulation of adrenal DNA synthesis in cadmium-treated male rats

    SciTech Connect

    Nishiyama, S.; Nakamura, K.

    1984-07-01

    Cadmium chloride (CdCl2) at a dose of 1 mg/kg body wt was injected into male rats of the Wistar strain, weighing 250 g on the average, twice a day (12-hr intervals) for 7 consecutive days. DNA and RNA contents and (/sup 3/H)-thymidine and (/sup 3/H)-uridine incorporation into the acid-insoluble fraction significantly increased in the adrenals of rats treated with Cd for 2 and 7 consecutive days. Adrenal protein content and weight also significantly increased. These results indicate that continued treatment with Cd stimulates DNA and RNA synthesis in the adrenal cortex, which in turn results in the increase of the total protein contents of the adrenal gland and subsequently in the enlargement of the gland. Serum adrenocorticotrophin (ACTH) and insulin levels in Cd-treated rats were not higher than control levels, suggesting that the stimulation of DNA synthesis in the adrenals of Cd-treated rats is due to factor(s) other than serum ACTH and insulin. Treatment with Cd inhibited DNA synthesis in cultured adrenocortical cells at concentrations of 10(-4) to 10(-8) M, suggesting that Cd does not directly stimulate DNA synthesis in the adrenal gland in vivo. Although the adrenal gland became enlarged, the total adrenal corticosterone content decreased significantly. The decrease of total adrenal corticosterone content may be due to the fall in serum ACTH level of Cd-treated rats.

  16. Relationship between in vitro growth of bovine oocytes and steroidogenesis of granulosa cells cultured in medium supplemented with bone morphogenetic protein-4 and follicle stimulating hormone.

    PubMed

    Sakaguchi, Kenichiro; Huang, Weiping; Yang, Yinghua; Yanagawa, Yojiro; Nagano, Masashi

    2017-07-15

    Bone morphogenetic protein-4 (BMP-4) and FSH play important regulatory roles in follicular growth and steroidogenesis in vivo. The purpose of this study was to investigate the effects of BMP-4 and FSH on in vitro growth (IVG) and steroidogenesis of bovine oocyte-cumulus-granulosa complexes (OCGCs). We cultured OCGCs collected from early antral follicles (0.5-1 mm) in medium without BMP-4 and FSH for 4 days and investigated the appearance of OCGCs and their steroidogenesis. During the first 4 days of IVG, morphologically normal OCGCs produced more estradiol-17β (E2), but less progesterone (P4). Morphologically normal OCGCs were subjected to an additional culture in medium supplemented with BMP-4 (0, 10, and 50 ng/mL) and FSH (0 and 0.5 ng/mL) until day 12. We examined the viability and steroidogenesis of OCGCs after 8 and 12 days of culture. Oocyte growth, characteristics of granulosa cells, and the maturational competence of oocytes were also investigated. On day 8, the viability of OCGCs cultured without FSH was higher in the 10 ng/mL BMP-4 group than in the 50 ng/mL BMP-4 group (P < 0.05). No significant difference was observed in the viability of groups cultured with FSH, regardless of the addition of BMP-4, and FSH improved the viability of 50 ng/mL BMP-4 group similar to 10 ng/mL BMP-4 group. The total number of granulosa cells was larger in 10 ng/mL BMP-4 group cultured with FSH than in 50 ng/mL BMP-4 group cultured with FSH on day 8 (P < 0.05). E2 production decreased from days 8-12, and P4 production increased throughout IVG culture, regardless of the addition of BMP-4 and FSH (P < 0.05). No significant differences in E2 production were observed between groups from days 4-8, regardless of whether BMP-4 was added without FSH; however, E2 production in the group cultured with 50 ng/mL BMP-4 was suppressed by FSH. BMP-4 suppressed E2 production from days 8-12, regardless of whether FSH was added. The group cultured with 10 ng/mL BMP-4

  17. Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality.

    PubMed

    Ahumada, C J; Salvador, I; Cebrian-Serrano, A; Lopera, R; Silvestre, M A

    2013-03-01

    < 0.05). The experimental group did not affect the total number of cells per blastocyst. In conclusion, this study showed that supplementation of the CM with EGF and IGF could partially avoid the deleterious effect of in vitro culture of small groups of bovine embryos, increasing blastocyst rates and decreasing apoptosis rates of these blastocysts.

  18. Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia.

    PubMed

    Tischler, A S; DeLellis, R A; Perlman, R L; Allen, J M; Costopoulos, D; Lee, Y C; Nunnemacher, G; Wolfe, H J; Bloom, S R

    1985-10-01

    Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.

  19. Adrenal Insufficiency and Addison's Disease

    MedlinePlus

    ... cortisol. If ACTH output is too low, cortisol production drops. Eventually, the adrenal glands can shrink due ... sodium and potassium in the blood. When aldosterone production falls too low, the body loses too much ...

  20. [Pheochromocytomas as adrenal gland incidentalomas].

    PubMed

    Cerović, Snezana; Cizmić, Milica; Milović, Novak; Ajdinović, Boris; Brajusković, Goran

    2002-07-01

    Adrenal incidentalomas are a heterogeneous group of pathological entities, including benign or malignant adrenocortical or medullary tumors, hormonally active or inactive lesions, which are identified incidentally during the examination of nonadrenal-related abdominal complaints. About 1.5% to 23% of adrenal incidentalomas are pheochromocytomas. Composite pheochromocytoma is a rare tumour of adrenal medulla with divergente clinical course. This type of pheochromocytoma is designated "composite" or "mixed," depending on whether pheochromocytoma and nonpheochromocytoma components show the same embryologic origin. Nonpheochromocytoma components found in the composite pheochromocytoma include ganglioneuroma, ganglioneuroblastoma, neuroblastoma, and malignant schwannoma. The biologic behavior of composite pheochromocytomas may be as difficult to predict as more traditional pheochromocytomas; based on the number of cases reported to date the presence of areas resembling ganglioneuroblastoma or neuroblastoma does not necessary indicate a poor prognosis. Some may behave in a malignant fashion with metastasis by a component of the tumour which has neural features. Pheochromocytomas and paragangliomas are well-defined entities. Some of their nonsporadic associations and unusual morphological appearances are not universally appreciated. We report on a rare association of left adrenal CP, with typical right adrenal phochromocytoma and retroperitoneal paraganglioma, and a review of literature. We analyzed the clinical and immunohistochemical features in a 24-year-old woman with composite pheochromocytoma localized in the left adrenal gland and associated with blood pressure of 200/140 mmHg. Abdominal computed tomography and 131-J MIBG revealed a 65 x 60 mm mass in the right adrenal gland, but no revealed 45 x 40 mm retroperitoneal mass and 20 x 20 mm mass in the left adrenal region. Serum and urinary adrenaline levels were high, and catecholamine levels in the blood sample of

  1. mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

    PubMed

    Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

    2016-08-15

    The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation

  2. In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

    PubMed

    Araújo, V R; Gastal, M O; Wischral, A; Figueiredo, J R; Gastal, E L

    2014-12-01

    The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 μL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Budesonide-related adrenal insufficiency.

    PubMed

    Arntzenius, Alexander; van Galen, Louise

    2015-10-01

    Iatrogenic adrenal insufficiency is a potential harmful side effect of treatment with corticosteroids. It manifests itself when an insufficient cortisol response to biological stress leads to an Addisonian crisis: a life-threatening situation. We describe a case of a patient who developed an Addisonian crisis after inappropriate discontinuation of budesonide (a topical steroid used in Crohn's disease) treatment. Iatrogenic adrenal insufficiency due to budesonide use has been rarely reported. Prescribers should be aware of the resulting risk for an Addisonian crisis.

  4. [Congenital Adrenal Hyperplasia in Adults].

    PubMed

    Vrbíková, Jana

    2016-01-01

    Congenital adrenal hyperplasia is a life-long disease requiring an integrated therapy. It may negatively influence the quality of life. In childhood, the main problems of the care of these patients involve sex determination and ensuring optimum growth and puberty. The therapeutic goals for adults are the prevention of Addisonian crisis and ensuring the best possible quality of life, including fertility.Key words: androgens - cardiovascular risk - congenital adrenal hyperplasia - bone density - testicular rest tumors.

  5. Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

    PubMed

    Romanov, Yu A; Balashova, E E; Volgina, N E; Kabaeva, N V; Dugina, T N; Sukhikh, G T

    2017-02-01

    Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

  6. Fibroblast growth factor-23 regulates parathyroid hormone and 1alpha-hydroxylase expression in cultured bovine parathyroid cells.

    PubMed

    Krajisnik, Tijana; Björklund, Peyman; Marsell, Richard; Ljunggren, Osten; Akerström, Göran; Jonsson, Kenneth B; Westin, Gunnar; Larsson, Tobias E

    2007-10-01

    Fibroblast growth factor-23 (FGF23) is a circulating factor that decreases serum levels of inorganic phosphate (Pi) as well as 1,25-dihydroxyvitamin D(3). Recent studies also suggest a correlation between serum levels of FGF23 and parathyroid hormone (PTH) in patients with chronic kidney disease. It is, however, unknown whether FGF23 directly modulates PTH expression, or whether the correlation is secondary to abnormalities in Pi and vitamin D metabolism. The objective of the current study was therefore to elucidate possible direct effects of FGF23 on bovine parathyroid cells in vitro. Treatment of parathyroid cells with a stabilized form of recombinant FGF23 (FGF23(R176Q)) induced a rise in early response gene-1 mRNA transcripts, a marker of FGF23 signaling. FGF23(R176Q) potently and dose-dependently decreased the PTH mRNA level within 12 h. In agreement, FGF23(R176Q) also decreased PTH secretion into conditioned media. In contrast, FGF23(R176Q) dose-dependently increased 1alpha-hydroxylase expression within 3 h. FGF23 (R176Q) did not affect cell viability nor induce apoptosis, whereas a small but significant increase in cell proliferation was found. We conclude that FGF23 is a negative regulator of PTH mRNA expression and secretion in vitro. Our data suggest that FGF23 may be a physiologically relevant regulator of PTH. This defines a novel function of FGF23 in addition to the previously established roles in controlling vitamin D and Pi metabolism.

  7. Proinflammatory responses of a hTERT-transformed, immortalized line of cultured bovine mammary epithelial cells (BME)

    USDA-ARS?s Scientific Manuscript database

    Primary cultures BME were generated from healthy mammary glands as described (Vet Immunol Immunopath 101(3-4):191-202, 2004). Towards immortalization, BME from four cows were pooled and transfected with pCI neo-hEST2-HA , a human telomerase segment containing a neomycin/Geneticin resistance select...

  8. Hypoxia modulates the barrier and coagulant function of cultured bovine endothelium. Increased monolayer permeability and induction of procoagulant properties.

    PubMed Central

    Ogawa, S; Gerlach, H; Esposito, C; Pasagian-Macaulay, A; Brett, J; Stern, D

    1990-01-01

    Exposure of cultured endothelium to environments with low concentrations of oxygen, in the range of those observed in pathophysiologic hypoxemic states in vivo, compromises cellular barrier and coagulant function. An atmosphere with PO2 approximately 14 mm Hg was not lethally toxic to endothelial cultures, but cells became larger and exhibited small intercellular gaps. At low oxygen concentrations, passage of macromolecular tracers through hypoxic endothelial monolayers was accelerated in a time- and dose-dependent manner, presumably by a paracellular pathway via the gaps. Cell surface coagulant properties of the endothelium were also perturbed. At PO2 approximately 14 mm Hg thrombomodulin antigen and functional activity on the cell surface were diminished by 80-90%, and Northern blots demonstrated suppression of thrombomodulin mRNA. The decrease in thrombomodulin was twice as great compared with the general decline in total protein synthesis in hypoxia. In addition, expression of a direct Factor X activator developed under hypoxic conditions; the activator was membrane-associated and expressed on the surface of intact cultures, Ca-dependent, inhibited by HgCl2 but not PMSF, and had Km approximately 25 micrograms/ml for the substrate at pH 7.4. Synthesis of the activator was blocked by inclusion of cycloheximide, but not warfarin, in the culture medium. These results demonstrate that endothelial function is perturbed in a selective manner in the presence of low concentrations of oxygen, providing insights into mechanisms which may contribute to vascular dysfunction in hypoxemic states. Images PMID:2156893

  9. Mitochondrial "movement" and lens optics following oxidative stress from UV-B irradiation: cultured bovine lenses and human retinal pigment epithelial cells (ARPE-19) as examples.

    PubMed

    Bantseev, Vladimir; Youn, Hyun-Yi

    2006-12-01

    Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV-B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-19 cells were irradiated with broadband UV-B radiation at energy levels of 0.5 and 1.0 J/cm(2). Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium-neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45 degrees on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria-specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta

  10. Revisiting bovine pyometra--new insights into the disease using a culture-independent deep sequencing approach.

    PubMed

    Knudsen, Lif Rødtness Vesterby; Karstrup, Cecilia Christensen; Pedersen, Hanne Gervi; Agerholm, Jørgen Steen; Jensen, Tim Kåre; Klitgaard, Kirstine

    2015-02-25

    The bacteria present in the uterus during pyometra have previously been studied using bacteriological culturing. These studies identified Fusobacterium necrophorum and Trueperella pyogenes as the major contributors to the pathogenesis of pyometra. However, an increasing number of culture-independent studies have demonstrated that the bacterial diversity in most environments is underestimated in culture-based studies. Consequently, fastidious pyometra-associated pathogens may have been overlooked. Therefore, the primary purpose of this study was to investigate the diversity of bacteria in the uterus of cows with pyometra by using culture-independent 16S rRNA PCR combined with next generation sequencing. We investigated the microbial composition in the uterus of 21 cows with pyometra, which were obtained from a Danish slaughterhouse. Similar to the observations from the culture studies, Fusobacteriaceae, the family that F. necrophorum belongs to, was the operational taxonomic unit (OTU) observed in the largest quantities. By contrast, the Actinomycetaceae family, which includes T. pyogenes, constituted only 1% of the total number of reads. Thus we cannot confirm the previously reported role of species from this family in the pathogenesis of pyometra. Finally, we identified a large number of sequences representing three families of Gram-negative bacteria in the pyometra samples: Porphyromonadaceae, Mycoplasmataceae, and Pasteurellaceae. It is likely that these families comprise potential pathogenic species of a fastidious nature, which have been overlooked in previous studies. Our results increase the knowledge of the complexity of the pyometra microbiota and suggest that pathogens in addition to F. necrophorum may be involved in the pathogenesis of pyometra.

  11. Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods.

    PubMed

    Araújo, V R; Gastal, M O; Wischral, A; Figueiredo, J R; Gastal, E L

    2015-10-01

    Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM(+) or TCM-199(+) as base media. The medium replacement methods were: Conventional - removal and subsequent addition of the same amount (60μl) in a 100μl aliquot (MEM-C and TCM-C), and Small Supplementation - addition of 5μl of fresh medium to an initial small aliquot (50μl), resulting in a final volume of 125μl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5°C in 5% CO2 and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P<0.01) follicular diameter, greater (P<0.02) growth rate, greater (P<0.02) antrum formation, as well as greater (P<0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P>0.05) the follicular and estradiol end points for TCM-199(+). The expression of the FSHR gene was greater (P<0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P<0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P<0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM(+) can be used to replace TCM-199(+) for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.

  12. Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing.

    PubMed

    Taponen, S; Salmikivi, L; Simojoki, H; Koskinen, M T; Pyörälä, S

    2009-06-01

    In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The "no-growth" samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10(3) to 10(7) bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-beta-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such

  13. AMPKα, C/EBPβ, CPT1β, GPR43, PPARγ, and SCD Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid

    PubMed Central

    Choi, S. H.; Park, S. K.; Johnson, B. J.; Chung, K. Y.; Choi, C. W.; Kim, K. H.; Kim, W. Y.; Smith, B.

    2015-01-01

    We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC) and intramuscular preadipocytes (IPA) were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco’s Modified Eagle Medium (DMEM) and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC) or 5% FBS/DMEM (IPA) with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001) carnitine palmitoyltransferase-1 beta (CPT1β) gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases). Oleic and linoleic acid decreased (p = 0.001) stearoyl-CoA desaturase (SCD) gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism. PMID:25656188

  14. AMPKα, C/EBPβ, CPT1β, GPR43, PPARγ, and SCD Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid.

    PubMed

    Choi, S H; Park, S K; Johnson, B J; Chung, K Y; Choi, C W; Kim, K H; Kim, W Y; Smith, B

    2015-03-01

    We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC) and intramuscular preadipocytes (IPA) were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's Modified Eagle Medium (DMEM) and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC) or 5% FBS/DMEM (IPA) with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001) carnitine palmitoyltransferase-1 beta (CPT1β) gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases). Oleic and linoleic acid decreased (p = 0.001) stearoyl-CoA desaturase (SCD) gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

  15. Compensatory adrenal growth - A neurally mediated reflex

    NASA Technical Reports Server (NTRS)

    Dallman, M. F.; Engeland, W. C.; Shinsako, J.

    1976-01-01

    The responses of young rats to left adrenalectomy or left adrenal manipulation were compared to surgical sham adrenalectomy in which adrenals were observed but not touched. At 12 h right adrenal wet weight, dry weight, DNA, RNA, and protein content were increased (P less than 0.05) after the first two operations. Left adrenal manipulation resulted in increased right adrenal weight at 12 h but no change in left adrenal weight. Sequential manipulation of the left adrenal at time 0 and the right adrenal at 12 h resulted in an enlarged right adrenal at 12 h (P less than 0.01), and an enlarged left adrenal at 24 h (P less than 0.05), showing that the manipulated gland was capable of response. Bilateral adrenal manipulation of the adrenal glands resulted in bilateral enlargement of 12 h (P less than 0.01). Taken together with previous results, these findings strongly suggest that compensatory adrenal growth is a neurally mediated reflex.

  16. Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods.

    PubMed

    van der Valk, J; Brunner, D; De Smet, K; Fex Svenningsen, A; Honegger, P; Knudsen, L E; Lindl, T; Noraberg, J; Price, A; Scarino, M L; Gstraunthaler, G

    2010-06-01

    Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, 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 and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media.

  17. Developmental and functional biology of the primate fetal adrenal cortex.

    PubMed

    Mesiano, S; Jaffe, R B

    1997-06-01

    The unique characteristics of the primate (particularly human) fetal adrenal were first realized in the early 1900s when its morphology was examined in detail and compared with that of other species. The unusual architecture of the human fetal adrenal cortex, with its unique and disproportionately enlarged fetal zone, its compact definitive zone, and its dramatic remodeling soon after birth captured the interest of developmental anatomists. Many detailed anatomical studies describing the morphology of the developing human fetal adrenal were reported between 1920 and 1960, and these morphological descriptions have not changed significantly. More recently, it has become clear that fetal adrenal cortical growth involves cellular hypertrophy, hyperplasia, apoptosis, and migration and is best described by the migration theory, i.e. cells proliferate in the periphery, migrate centripetally, differentiate during their migration to form the functional cortical zones, and then likely undergo apoptosis in the center of the cortex. Consistent with this model, cells of intermediate phenotype, arranged in columnar cords typical of migration, have been identified between the definitive and fetal zones. This cortical area has been referred to as the transitional zone and, based on the expression of steroidogenic enzymes, we consider it to be a functionally distinct cortical zone. Elegant experiments during the 1950s and 1960s demonstrated the central role of the primate fetal adrenal cortex in establishing the estrogenic milieu of pregnancy. Those findings were among the first indications of the function and physiological role of the human fetal adrenal cortex and led Diczfalusy and co-workers to propose the concept of the feto-placental unit, in which DHEA-S produced by the fetal adrenal cortex is used by the placenta for estrogen synthesis. Tissue and cell culture techniques, together with improved steroid assays, revealed that the fetal zone is the primary source of DHEA

  18. Cushing syndrome due to adrenal tumor

    MedlinePlus

    ... syndrome. It occurs when a tumor of the adrenal gland releases excess amounts of the hormone cortisol. Causes ... hormone cortisol. This hormone is made in the adrenal glands . Too much cortisol can be due to various ...

  19. [Addison's disease, primary adrenal insufficiency in adults].

    PubMed

    Krikke, Maaike; ten Wolde, Marije; Smit, Natalie

    2013-01-01

    Adrenal insufficiency is a rare but fatal disease if left unrecognized. Symptoms often mimic more prevalent diseases. We discuss three patients with primary adrenal insufficiency. These cases illustrate that presenting symptoms such as syncope, nausea, vomiting, weight loss and hypoglycemia are often non-specific and, therefore, often not immediately recognized. When an adrenal crisis is suspected, glucocorticoids should be given promptly. The symptoms are caused by insufficient production of adrenal hormones due to destruction of the adrenal glands by auto-immune adrenalitis. An ACTH stimulation test should confirm the diagnosis when primary adrenal insufficiency is suspected. Treatment consists of glucocorticoid and mineralocorticoid replacement. Primary adrenal insufficiency is a 'master of disguise'. Unexplained syncope, vomiting, weight loss or hypoglycemia should prompt suspicion of this disease.

  20. Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development.

    PubMed

    Kropp, Jenna; Khatib, Hasan

    2015-09-01

    Dairy cattle fertility has declined over time due to factors including reduced fertilization and early embryonic loss. To counter fertility problems and better study preimplantation embryonic development, in vitro production systems have been developed. These systems largely assess embryos based on their morphology, which is not a strong indicator of developmental potential. Currently, no biomarkers can be used to noninvasively survey an embryo's potential in terms of its development and ability to establish a pregnancy. Thus, the objective of this study was to characterize and identify microRNA (miRNA) in culture media of embryos of differing developmental competence for future development as noninvasive biomarkers of embryo quality. The MiRNA sequencing of media conditioned by blastocyst and degenerate (those that failed to develop from the morula to blastocyst stage) embryos, revealed 11 differentially expressed miRNA; all were higher in concentration in degenerate conditioned media. Differential expression of mature microRNA (miR)-24, miR-191, and miR-148a was further validated using quantitative real-time PCR. Functional analysis of miR-24 revealed that addition of a mimic miRNA to culture media of morulae embryos resulted in a 27.3% decrease in development to the blastocyst stage. Furthermore, expression of miR-24 was 44.29-fold higher in blastocysts cultured with a miR-24 mimic compared with control blastocysts. Interestingly, the expression of CDKN1b, a target gene of miR-24 was repressed in embryos grown in the presence of the miRNA mimic. Mimic supplementation experiments suggest that miRNA are taken up by the embryo and that extracellular miRNA affect embryonic development. Overall, identification of a rich extracellular milieu in conditioned media sets the framework for future studies to determine the long-term predictive ability of embryo-based miRNA biomarkers on pregnancy outcome. Copyright © 2015 American Dairy Science Association. Published by

  1. Development and de novo protein synthetic activity of bovine embryos produced in vitro in different culture systems.

    PubMed

    Kuran, M; Robinson, J J; Staines, M E; McEvoy, T G

    2001-01-15

    In vitro matured (IVM) and fertilized (IVF) putative Day 1 zygotes (Day 0 = IVF) were allocated randomly to culture in formulations based on Synthetic Oviduct Fluid (SOF) medium and identified on the basis of their contrasting principal supplements, which were 10% v/v steer serum (SS; n = 558) or 4 mg/mL crystalline BSA (SBSA; n = 531) or 3 mg/mL polyvinyl alcohol (SPVA; n = 607) in 9 replicates. SBSA and SPVA also contained 10 microg/mL non-essential amino acids, while the former was further supplemented with 20 microL/mL essential amino acids and the latter with 0.5 mmol/L sodium citrate and 5 ng/mL epidermal growth factor. Zygotes were cultured in 20 microL drops (4 zygotes per drop) until Day 8 in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 39 degrees C and droplets were renewed every 48 hours. The incidence of zygote cleavage was lower (P < 0.05) in SS (mean +/- SEM = 61 +/- 3%) than in SBSA (76 +/- 3%) but not in SPVA (72 +/- 4%) up to Day 3. The SPVA generated a lower yield of blastocysts on Day 7 (12 +/- 2%; P < 0.001) and by Day 8 (21 +/- 4%; P < 0.01) than did SS (33 +/- 3%; 40 +/- 3%) and SBSA (30 +/- 3%; 37 +/- 4%). Cell numbers (n) and diameters (d) of blastocysts on Day 8 were greater (P < 0.001; Replicates 1 to 5) in embryos from SBSA (n, 156 +/- 9; d, 203 +/- 4 microm) than in those from SS (n, 81 +/- 4; d, 177 +/- 3 microm) and SPVA (n, 76 +/- 5; d, 167 +/- 3 microm). Embryos produced in SS incorporated less 3H-phenylalanine into PCA-precipitable protein (replicates 6 to 9; log10 dpm = 3.03 +/- 0.04) than did embryos cultured in SBSA (3.21 +/- 0.03; P < 0.001) or in SPVA (3.14 +/- 0.03; NS). In conclusion, blastocyst yield was poor in SPVA, but the embryos had metabolic activities similar to those of embryos produced in SBSA. Blastocyst yields from SS were not compromised but their capacity for de novo protein synthesis was reduced significantly.

  2. Adrenal cortex dysfunction: CT findings

    SciTech Connect

    Huebener, K.H.; Treugut, H.

    1984-01-01

    The computed tomographic appearance of the adrenal gland was studied in 302 patients with possible endocrinologic disease and 107 patients undergoing CT for nonendocrinologic reasons. Measurements of adrenal size were also made in 100 adults with no known adrenal pathology. CT proved to be a sensitive diagnostic tool in combination with clinical studies. When blood hormone levels are increased, CT can differentiate among homogeneous organic hyperplasia, nodular hyperplasia, benign adenoma, and malignant cortical adenoma. When blood hormone levels are decreased, CT can demonstrate hypoplasia or metastatic tumorous destruction. Calcifications can be demonstrated earlier than on plain radiographs. When hormone elimination is increased, the morphologic substrate can be identified; tumorous changes can be localized and infiltration of surrounding organs recognized.

  3. Adrenal gland disease in ferrets.

    PubMed

    Simone-Freilicher, Elisabeth

    2008-01-01

    Adrenal gland disease in ferrets is unique to this species, with clinical signs and pathophysiology different from those seen in the dog. Its prevalence is increasing; 70% of pet ferrets in the United States were affected in 2003. The exact causes of the adrenal gland changes that lead to the disease are not known. Early oophorohysterectomies and neutering, combined with the artificially prolonged photoperiod experienced by indoor pet ferrets, and a possible genetic component, may be contributing factors. Signs of adrenal gland disease include progressive hair loss, pruritus, lethargy, atrophy, and, in female ferrets, vulvar swelling. An understanding of the signs and physiologic changes is necessary for diagnosis and treatment. A review of anatomy, physiology, and current surgical and medical options is presented.

  4. Adrenal myelolipoma with osseous metaplasia and hypercortisolism

    PubMed Central

    Kumar, Ujwal; Priyadarshi, Shivam; Tomar, Vinay; Vohra, Rishi Raj

    2017-01-01

    Adrenal myelolipomas are rare adrenal tumors generally diagnosed incidentally. A 42-year-old female reported to us with complaints of left flank pain attributable to her left ureteric calculi. On evaluation, a large adrenal mass was diagnosed along with hypercortisolism. After adrenalectomy, the histopathology revealed adrenal myelolipoma along with osseous metaplasia not reported in English literature, to the best of our knowledge till date. PMID:28216934

  5. Chlorpyrifos alters functional integrity and structure of an in vitro BBB model: co-cultures of bovine endothelial cells and neonatal rat astrocytes.

    PubMed

    Parran, Damani K; Magnin, Geraldine; Li, Wen; Jortner, Bernard S; Ehrich, Marion

    2005-01-01

    The blood-brain barrier (BBB) is a structural and functional interface between the circulatory system and the brain. Organophosphorous compounds such as chlorpyrifos (CPF) may cross the BBB and disrupt BBB integrity and function. To determine events that may contribute to CPF toxicity, we used an in vitro BBB model in which bovine microvascular endothelial cells (BMEC) and neonatal rat astrocytes were co-cultured. We hypothesized that CPF is metabolized by the BBB leading to an inhibition of esterase activity and a disruption of the BBB. The co-culturing of BMECs and astrocytes resulted in tight junction formation as determined by electron microscopy, electrical resistance and western blot analysis of two tight junction-associated proteins (ZO-1 and e-cadherin). We observed time dependent increases in ZO-1 and e-cadherin expression and electrical resistance during BBB formation, which were maximal after 9-13 days of co-culturing. The CPF concentration and production of its metabolites were monitored by HPLC following 24 h exposure to CPF on the luminal side of the BBB. We found that the BBB metabolized CPF, with the metabolite 2,3,6-trichloro-2-pyridinol being the major product. CPF and its metabolites were detected on the abluminal side of the BBB suggesting that CPF crossed this barrier. CPF was also detected intracellularly and on the membrane inserts. At tested concentrations (0.1-10 microM), CPF inhibited both carboxylesterase (CaE) and cholinesterase (ChE) activities in BMECs by 43-100%, while CPF-oxon totally inhibited CaE and ChE activity in concentrations as low as 0.1 microM. CPF also caused a concentration-dependent decrease in electrical resistance, with significant inhibition observed at 1 nM and complete loss at 1 microM. These data show that low concentrations of CPF and its metabolites are present within the BBB. CPF and its metabolites, especially CPF-oxon, contribute to the inhibition of CaE and ChE activity, as well as the alteration of BBB

  6. ARMC5 mutations in macronodular adrenal hyperplasia with Cushing's syndrome.

    PubMed

    Assié, Guillaume; Libé, Rossella; Espiard, Stéphanie; Rizk-Rabin, Marthe; Guimier, Anne; Luscap, Windy; Barreau, Olivia; Lefèvre, Lucile; Sibony, Mathilde; Guignat, Laurence; Rodriguez, Stéphanie; Perlemoine, Karine; René-Corail, Fernande; Letourneur, Franck; Trabulsi, Bilal; Poussier, Alix; Chabbert-Buffet, Nathalie; Borson-Chazot, Françoise; Groussin, Lionel; Bertagna, Xavier; Stratakis, Constantine A; Ragazzon, Bruno; Bertherat, Jérôme

    2013-11-28

    Corticotropin-independent macronodular adrenal hyperplasia may be an incidental finding or it may be identified during evaluation for Cushing's syndrome. Reports of familial cases and the involvement of both adrenal glands suggest a genetic origin of this condition. We genotyped blood and tumor DNA obtained from 33 patients with corticotropin-independent macronodular adrenal hyperplasia (12 men and 21 women who were 30 to 73 years of age), using single-nucleotide polymorphism arrays, microsatellite markers, and whole-genome and Sanger sequencing. The effects of armadillo repeat containing 5 (ARMC5) inactivation and overexpression were tested in cell-culture models. The most frequent somatic chromosome alteration was loss of heterozygosity at 16p (in 8 of 33 patients for whom data were available [24%]). The most frequent mutation identified by means of whole-genome sequencing was in ARMC5, located at 16p11.2. ARMC5 mutations were detected in tumors obtained from 18 of 33 patients (55%). In all cases, both alleles of ARMC5 carried mutations: one germline and the other somatic. In 4 patients with a germline ARMC5 mutation, different nodules from the affected adrenals harbored different secondary ARMC5 alterations. Transcriptome-based classification of corticotropin-independent macronodular adrenal hyperplasia indicated that ARMC5 mutations influenced gene expression, since all cases with mutations clustered together. ARMC5 inactivation decreased steroidogenesis in vitro, and its overexpression altered cell survival. Some cases of corticotropin-independent macronodular adrenal hyperplasia appear to be genetic, most often with inactivating mutations of ARMC5, a putative tumor-suppressor gene. Genetic testing for this condition, which often has a long and insidious prediagnostic course, might result in earlier identification and better management. (Funded by Agence Nationale de la Recherche and others.).

  7. Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

    PubMed

    Zampieri, Dávila; Santos, Vanessa G; Braga, Patrícia A C; Ferreira, Christina R; Ballottin, Daniela; Tasic, Ljubica; Basso, Andréa C; Sanches, Bruno V; Pontes, José H F; da Silva, Bárbara Pereira; Garboggini, Fabiana Fantinatti; Eberlin, Marcos N; Tata, Alessandra

    2013-09-01

    Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Radionuclide therapy of adrenal tumors.

    PubMed

    Carrasquillo, Jorge A; Pandit-Taskar, Neeta; Chen, Clara C

    2012-10-01

    Adrenal tumors arising from chromaffin cells will often accumulate radiolabeled metaiodobenzylguanidine (MIBG) and thus are amenable to therapy with I-131 MIBG. More recently, therapy studies have targeted the somatostatin receptors using Lu-177 or Y-90 radiolabeled somatostatin analogs. Because pheochromocytoma (PHEO)/paraganglioma (PGL) and neuroblastoma (NB), which often arise from the adrenals, express these receptors, clinical trials have been performed with these reagents. We will review the experience using radionuclide therapy for targeting PHEO/PGL and NBs. Copyright © 2012 Wiley Periodicals, Inc.

  9. [Immunoendocrine associations in adrenal glands].

    PubMed

    Sterzl, I; Hrdá, P

    2010-12-01

    Immune and endocrine systems are basic regulatory mechanisms of organism and, including the nervous system, maintain the organism's homeostasis. The main immune system representatives are mononuclear cells, T- and B-cells and their products, in the endocrine system the main representatives are cells of the glands with inner secretion and their products. One of the most important glands for maintaining homeostasis are adrenal glands. It has been proven that either cells of the immune system, either endocrine cells can, although in trace amounts, produce mutually mediators of both systems (hormones, cytokines). Disorders in one system can lead to pathological symptoms in the other system. Also here represent adrenals an important model.

  10. In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

    PubMed

    Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige

    2015-07-01

    The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients.

  11. Spontaneous bilateral adrenal hemorrhage following cholecystectomy.

    PubMed

    Dahan, Meryl; Lim, Chetana; Salloum, Chady; Azoulay, Daniel

    2016-06-01

    Postoperative bilateral adrenal hemorrhage is a rare but potentially life-threatening complication. This diagnosis is often missed because the symptoms and laboratory results are usually nonspecific. We report a case of bilateral adrenal hemorrhage associated with acute primary adrenal insufficiency following laparoscopic cholecystectomy. The knowledge of this uncommon complication following any abdominal surgery allows timey diagnosis and rapid treatment.

  12. Spontaneous Retroperitoneal Hemorrhage from Adrenal Artery Aneurysm

    SciTech Connect

    Gonzalez Valverde, F.M. Balsalobre, M.; Torregrosa, N.; Molto, M.; Gomez Ramos, M.J.; Vazquez Rojas, J.L.

    2007-04-15

    Spontaneous adrenal hemorrhage is a very rare but serious disorder of the adrenal gland that can require emergent treatment. We report on a 42-year-old man who underwent selective angiography for diagnosis and treatment of retroperitoneal hemorrhage from small adrenal artery aneurysm. This case gives further details about the value of transluminal artery embolization in the management of visceral aneurysm rupture.

  13. The adrenal glands and their functions.

    PubMed

    De Silva, Deepthi C; Wijesiriwardene, Bandula

    2007-09-01

    The adrenal glands secrete hormones essential for metabolism, regulation of blood pressure, and sodium and glucose homeostasis. Hypo- or hypersecretion of these hormones is life threatening. Understanding the physiological functions of adrenal hormones is a prerequisite to the management of adrenal gland disease.

  14. Spontaneous bilateral adrenal hemorrhage following cholecystectomy

    PubMed Central

    Dahan, Meryl; Lim, Chetana; Salloum, Chady

    2016-01-01

    Postoperative bilateral adrenal hemorrhage is a rare but potentially life-threatening complication. This diagnosis is often missed because the symptoms and laboratory results are usually nonspecific. We report a case of bilateral adrenal hemorrhage associated with acute primary adrenal insufficiency following laparoscopic cholecystectomy. The knowledge of this uncommon complication following any abdominal surgery allows timey diagnosis and rapid treatment. PMID:27275469

  15. Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension.

    PubMed

    Jorssen, Ellen P A; Langbeen, An; Fransen, Erik; Martinez, Emilia L; Leroy, Jo L M R; Bols, Peter E J

    2014-08-01

    The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O2) or high (20% O2) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO2, and 5% or 20% O2. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was

  16. Qualitative and quantitative impacts assessment of contagious bovine pleuropneumonia in Fulani pastoral herds of North-central Nigeria: The associated socio-cultural factors.

    PubMed

    Alhaji, N B; Babalobi, O O

    2016-06-01

    Contagious bovine pleuropneumonia is one of the most important trans-boundary disease affecting Fulani cattle herds of Nigeria and whose control is urgently needed. A Participatory Epidemiology approach and cross-sectional study were concurrently conducted to investigate qualitative and quantitative impacts of CBPP, respectively and associated socio-cultural factors that influenced exposure of Fulani nomadic pastoral communities to its risk in Niger State, North-central Nigeria between January and December 2013. A total of nine pastoral communities were purposively selected for qualitative impact assessment using Participatory Rural Appraisal tools, while 765 cattle randomly sampled from 125 purposively selected nomadic herds were analyzed using c-ELISA. Data on socio-cultural characteristics were collected using structured questionnaires administered on nomadic herd owners of the 125 selected herds. Kendall's Coefficient of Concordance W statistics and OpenEpi 2.3 were used for statistical analyses. Pastoralists' dependent factors associated with their socio-cultural activities were tested using Chisquare tests and likelihood backward logistic regressions. The mean proportional piles (relative qualitative impact) of CBPP was 12.6%, and nomads agreement on this impact was strong (W=0.6855) and statistically significant (P<0.001). This was validated by 16.2% (95% CI: 13.7, 19.0) sero-positive (quantitative impact). Highest sero-prevalence of 25.3% was observed in Northern agro-ecological zone, while lowest of 6.2% was in Eastern zone. Pastoralists in the age groups 51-60 and 61-70 years were more likely (OR 13.07; 95% CI: 3.21, 53.12 and OR 7.10; 95% CI: 1.77, 28.33, respectively) to have satisfactory information/awareness on CBPP and lowland transhumance pastoralists were more likely (OR 5.21; 95% CI: 2.01, 13.54) to have satisfactory information. Socio-cultural activities of extensive husbandry system was six times more likely (OR 5.79; 95% CI: 2.55, 13.13) to be

  17. Assessment of a bovine co-culture, scaffold-free method for growing meniscus-shaped constructs.

    PubMed

    Aufderheide, Adam C; Athanasiou, Kyriacos A

    2007-09-01

    Using a self-assembly (SA), scaffoldless method, five high-density co-cultures with varied ratios of meniscal fibrochondrocytes (MFCs) and articular chondrocytes (ACs) were seeded into novel meniscus-specific, ring-shaped agarose wells. The following ratios of MFCs to ACs were used: 0% MFC, 25% MFC, 50% MFC, 75% MFC, and 100% MFC. Over 4 weeks, all ratios of cells self-assembled into three-dimensional constructs with varying mechanobiological and morphological properties. All groups stained for collagen II (Col II), and all groups except the 0% MFC group stained for collagen I (Col I). It was found that the tensile modulus was proportional to the percentage of MFCs employed. The 100% MFC group yielded the greatest mechanical stiffness with 432.2 +/- 47 kPa tensile modulus and an ultimate tensile strength of 23.7 +/- 2.4 kPa. On gross inspection, the 50% MFC constructs were the most similar to our idealized meniscus shape, our primary criterion. A second experiment was performed to examine the anisotropy of constructs as well as to directly compare the scaffoldless, SA method with a poly-glycolic acid (PGA) scaffold-based construct. When compared to PGA constructs, the SA groups were 2-4 times stiffer and stronger in tension. Further, at 8 weeks, SA groups exhibited circumferential fiber bundles similar to native tissue. When pulled in the circumferential direction, the SA group had significantly higher tensile modulus (226 +/- 76 kPa) than when pulled in the radial direction (67 +/- 32 kPa). The PGA constructs had neither a directional collagen fiber orientation nor differences in mechanical properties in the radial or circumferential direction. It is suggested that the geometric constraint imposed by the ring-shaped, nonadhesive mold guides collagen fibril directionality and, thus, alters mechanical properties. Co-culturing ACs and MFCs in this manner appears to be a promising new method for tissue engineering fibrocartilaginous tissues exhibiting a spectrum of

  18. [Metabolic correction of structural changes in adrenal glands during experimental widespread purulent peritonitis].

    PubMed

    Kosinets, V A; Fedotov, D N

    2012-01-01

    Experiments on 55 male chinchilla rabbits with model widespread purulent peritonitis have been performed for determinig structural changes in adrenal glands with the aid of optical microscopy. The introduction of aerobic-anaerobic culture of E. Coli and B. Fragilis into the abdominal cavity causes expressed structural changes in parenchyma of adrenal glands within 6 hours. It is established for the first time that the administration of metabolic drugs citoflavin (containing succinic acid) and neoton (containing creatine phosphate) prevents the development of pathological structural changes in adrenal glands under conditions of experimental widespread purulent peritonitis.

  19. Transport of monocarboxylic acids at the blood-brain barrier: Studies with monolayers of primary cultured bovine brain capillary endothelial cells

    SciTech Connect

    Terasaki, T.; Takakuwa, S.; Moritani, S.; Tsuji, A. )

    1991-09-01

    The kinetics and mechanism of the transport of monocarboxylic acids (MCAs) were studied by using primary cultured bovine brain capillary endothelial cells. Concentration-dependent uptake of acetic acid was observed, and the kinetic parameters were estimated as follows: the Michaelis constant, Kt, was 3.41 {plus minus} 1.87 mM, the maximum uptake rate, Jmax, was 144.7 {plus minus} 55.7 nmol/mg of protein/min and the nonsaturable first-order rate constant, Kd, was 6.66 {plus minus} 1.98 microliters/mg of protein/min. At medium pH below 7.0, the uptake rate of (3H)acetic acid increased markedly with decreasing medium pH, whereas pH-independent uptake was observed in the presence of 10 mM acetic acid. An energy requirement for (3H)acetic acid uptake was also demonstrated, because metabolic inhibitors (2,4-dinitrophenol and rotenone) reduced significantly the uptake rate (P less than .05). Carbonylcyanide-p-trifluoro-methoxyphenylhydrazone, a protonophore, inhibited significantly the uptake of (3H)acetic acid at medium pH of 5.0 and 6.0, whereas 4,4{prime}-diisothiocyanostilben-2,2{prime}-disulfonic acid did not. Several MCAs inhibited significantly the uptake rate of (3H)acetic acid, whereas di- and tricarboxylic acids did not. The uptake of (3H)acetic acid was competitively inhibited by salicylic acid, with an inhibition constant, Ki, of 3.60 mM, suggesting a common transport system between acetic acid and salicylic acid. Moreover, at the medium pH of 7.4, salicylic acid and valproic acid inhibited significantly the uptake of (3H)acetic acid, demonstrating that the transport of MCA drugs could also be ascribed to the MCA transport system at the physiologic pH.

  20. The influence of in vitro fertilization and embryo culture on the embryo epigenetic constituents and the possible consequences in the bovine model.

    PubMed

    Sirard, M-A

    2017-03-06

    Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.

  1. Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture.

    PubMed Central

    Schmitt, J; Keil, G M

    1996-01-01

    The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schönböken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1. The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication. Concomitant deletion of nine 3' codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins. The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa. Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids. A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA. The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions. In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture. PMID:8551568

  2. Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

    PubMed

    Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

    2014-01-01

    Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

  3. Adrenal Insufficiency and Addison's Disease

    MedlinePlus

    ... used if the diagnosis remains unclear. What other tests might a health care provider perform after diagnosis of adrenal insufficiency? After ... skin. A nurse or lab technician performs the test in a health care provider’s office; a patient does not need anesthesia. ...

  4. Anesthetic Considerations on Adrenal Gland Surgery

    PubMed Central

    Domi, Rudin; Sula, Hektor; Kaci, Myzafer; Paparisto, Sokol; Bodeci, Artan; Xhemali, Astrit

    2015-01-01

    Adrenal gland surgery needs a multidisciplinary team including endocrinologist, radiologist, anesthesiologist, and surgeon. The indications for adrenal gland surgery include hormonal secreting and non-hormonal secreting tumors. Adrenal hormonal secreting tumors present to the anesthesiologist unique challenges requiring good preoperative evaluation, perioperative hemodynamic control, corrections of all electrolytes and metabolic abnormalities, a detailed and careful anesthetic strategy, overall knowledge about the specific diseases, control and maintaining of postoperative adrenal function, and finally a good collaboration with other involved colleagues. This review will focus on the endocrine issues, as well as on the above-mentioned aspects of anesthetic management during hormone secreting adrenal gland tumor resection. PMID:25368694

  5. [Frequency of Kongenital Adrenal Hyperplasia (author's transl)].

    PubMed

    Müller, W; Prader, M; Kofler, J; Glatzl, J; Geir, W

    1979-01-01

    The frequency of homozygous congenital adrenal hyperplasia in Tyrol is found to be 1 : 8991, the gene-frequency for congenital adrenal hyperplasia 1 : 95 and the frequency of heterozygous congenital adrenal hyperplasia 1 : 48. Our data is compared on a numerical and statistical base with that in Zürich and Munich with regard to the frequency of congenital adrenal hyperplasia, to its distribution with and without salt loss and to its sex-distribution. According to our study one may assume a frequency of homozygous congenital adrenal hyperplasia in Tyrol, Zürich and Munich of 1 : 7000--10,000.

  6. The global effect of follicle-stimulating hormone and tumour necrosis factor α on gene expression in cultured bovine ovarian granulosa cells

    PubMed Central

    2014-01-01

    Background Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone; FSH) and inhibitory (tumour necrosis factor alpha; TNFα) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3–6. Initially dose–response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNFα (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNFα, or with FSH plus TNFα (n = 4 per group) and RNA was harvested for microarray analyses. Results Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNFα/TNFα plus FSH. The effect of TNFα on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNFα being similar to those of FSH plus TNFα treatment. TNFα treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST, INHA, INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNFα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons γ and

  7. Adrenal adrenoceptors in heart failure

    PubMed Central

    de Lucia, Claudio; Femminella, Grazia D.; Gambino, Giuseppina; Pagano, Gennaro; Allocca, Elena; Rengo, Carlo; Silvestri, Candida; Leosco, Dario; Ferrara, Nicola; Rengo, Giuseppe

    2014-01-01

    Heart failure (HF) is a chronic clinical syndrome characterized by the reduction in left ventricular (LV) function and it represents one of the most important causes of morbidity and mortality worldwide. Despite considerable advances in pharmacological treatment, HF represents a severe clinical and social burden. Sympathetic outflow, characterized by increased circulating catecholamines (CA) biosynthesis and secretion, is peculiar in HF and sympatholytic treatments (as β-blockers) are presently being used for the treatment of this disease. Adrenal gland secretes Epinephrine (80%) and Norepinephrine (20%) in response to acetylcholine stimulation of nicotinic cholinergic receptors on the chromaffin cell membranes. This process is regulated by adrenergic receptors (ARs): α2ARs inhibit CA release through coupling to inhibitory Gi-proteins, and β ARs (mainly β2ARs) stimulate CA release through coupling to stimulatory Gs-proteins. All ARs are G-protein-coupled receptors (GPCRs) and GPCR kinases (GRKs) regulate their signaling and function. Adrenal GRK2-mediated α2AR desensitization and downregulation are increased in HF and seem to be a fundamental regulator of CA secretion from the adrenal gland. Consequently, restoration of adrenal α2AR signaling through the inhibition of GRK2 is a fascinating sympatholytic therapeutic strategy for chronic HF. This strategy could have several significant advantages over existing HF pharmacotherapies minimizing side-effects on extra-cardiac tissues and reducing the chronic activation of the renin–angiotensin–aldosterone and endothelin systems. The role of adrenal ARs in regulation of sympathetic hyperactivity opens interesting perspectives in understanding HF pathophysiology and in the identification of new therapeutic targets. PMID:25071591

  8. Posterior retroperitoneoscopic approach to the adrenal arteries.

    PubMed

    Lotti, Marco; Giulii Capponi, Michela

    2016-12-01

    Differently from transperitoneal adrenalectomy, with the posterior retroperitoneoscopic approach adrenal arteries are dissected first [1, 2]. Knowledge of their position is pivotal as they are covered by peri-adrenal fat [3, 4]. Four posterior retroperitoneoscopic adrenalectomies were selected, in which adrenal arteries are dissected to show their path and how they can be localized among peri-adrenal fat. A video is presented herein, which focuses on surgical anatomy of adrenal arteries when approached during a posterior retroperitoneoscopic adrenalectomy. Details about relative positions between adrenal arteries and adjacent structures are considered and shown during their dissection. The posterior retroperitoneoscopic approach offers a direct view of adrenal arteries and allows for their exposure and safe division in the early steps of adrenalectomy.

  9. Cushing's syndrome, nodular adrenal hyperplasia and virilizing carcinoma.

    PubMed

    Anderson, D C; Child, D F; Sutcliffe, C H; Buckley, C H; Davies, D; Longson, D

    1978-07-01

    A 48-year-old hypertensive diabetic woman rapidly became virilized. Urine 17-oxo-and oxogenic steroids and plasma testosterone, androstenedione, DHEA, DHEA-sulphate and androstenediol were greatly elevated. Plasma cortisol was constantly high and was not suppressed by dexamethasone. Circulating immunoreactive ACTH was consistently detectable at 18-24 ng/l. A 450 g carcinoma arising from a nodular hyperplastic right adrenal gland was resected. Production by the tumour of 17a-hydroxypregnenolone, 17a-hydroxyprogesterone and five C-19 steroids, but very little prenenolone, progesterone or cortisol, was shown by blood sampling, tumour culture and dramatic falls after operation. The plasma cortisol fell to half, with no diurnal variation, consistent with persistent Cushing's syndrome, and the plasma ACTH rose to 55 ng/l. She died 3 months later from a myocardial infarction. Autopsy revealed a pituitary basophil adenoma at a site where radiologically there had been an indentation in the fossa floor for at least 7 years. The left adrenal gland showed nodular hyperplasia. Therefore we conclude that mild pituitary-dependent Cushing's syndrome may have been present for many years before development of a virilizing carcinoma. This case demonstrates that adrenal carcinoma in man can sometimes develop as a consequence of nodular adrenal hyperplasia which may in turn be due to long-standing trophic hyper-stimulation.

  10. Management of adolescents with congenital adrenal hyperplasia.

    PubMed

    Merke, Deborah P; Poppas, Dix P

    2013-12-01

    The management of congenital adrenal hyperplasia involves suppression of adrenal androgen production, in addition to treatment of adrenal insufficiency. Management of adolescents with congenital adrenal hyperplasia is especially challenging because changes in the hormonal milieu during puberty can lead to inadequate suppression of adrenal androgens, psychosocial issues often affect adherence to medical therapy, and sexual function plays a major part in adolescence and young adulthood. For these reasons, treatment regimen reassessment is indicated during adolescence. Patients with non-classic congenital adrenal hyperplasia require reassessment regarding the need for glucocorticoid drug treatment. No clinical trials have compared various regimens for classic congenital adrenal hyperplasia in adults, thus therapy is individualised and based on the prevention of adverse outcomes. Extensive patient education is key during transition from paediatric care to adult care and should include education of females with classic congenital adrenal hyperplasia regarding their genital anatomy and surgical history. Common issues for these patients include urinary incontinence, vaginal stenosis, clitoral pain, and cosmetic concerns; for males with classic congenital adrenal hyperplasia, common issues include testicular adrenal rest tumours. Transition from paediatric to adult care is most successful when phased over many years. Education of health-care providers on how to successfully transition patients is greatly needed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Management of adolescents with congenital adrenal hyperplasia

    PubMed Central

    Merke, Deborah P; Poppas, Dix P

    2014-01-01

    The management of congenital adrenal hyperplasia involves suppression of adrenal androgen production, in addition to treatment of adrenal insufficiency. Management of adolescents with congenital adrenal hyperplasia is especially challenging because changes in the hormonal milieu during puberty can lead to inadequate suppression of adrenal androgens, psychosocial issues often affect adherence to medical therapy, and sexual function plays a major part in adolescence and young adulthood. For these reasons, treatment regimen reassessment is indicated during adolescence. Patients with non-classic congenital adrenal hyperplasia require reassessment regarding the need for glucocorticoid drug treatment. No clinical trials have compared various regimens for classic congenital adrenal hyperplasia in adults, thus therapy is individualised and based on the prevention of adverse outcomes. Extensive patient education is key during transition from paediatric care to adult care and should include education of females with classic congenital adrenal hyperplasia regarding their genital anatomy and surgical history. Common issues for these patients include urinary incontinence, vaginal stenosis, clitoral pain, and cosmetic concerns; for males with classic congenital adrenal hyperplasia, common issues include testicular adrenal rest tumours. Transition from paediatric to adult care is most successful when phased over many years. Education of health-care providers on how to successfully transition patients is greatly needed. PMID:24622419

  12. Adrenal imaging (Part 1): Imaging techniques and primary cortical lesions

    PubMed Central

    Panda, Ananya; Das, Chandan J.; Dhamija, Ekta; Kumar, Rakesh; Gupta, A. K.

    2015-01-01

    Adrenal glands can be affected by a variety of lesions. Adrenal lesions can either be primary, of adrenal origin, or secondary to other pathologies. Primary adrenal lesions can further be either of cortical or medullary origin. Functioning adrenal lesions can also give clues to the histologic diagnosis and direct workup. Over the years, various imaging techniques have been developed that have increased diagnostic accuracy and helped in better characterization of adrenal lesions non-invasively. In the first part of the two part series, we review adrenal imaging techniques and adrenal cortical tumors such as adenomas, adrenocortical tumors, adrenal hyperplasia and oncocytomas. PMID:25593820

  13. Addiction and the adrenal cortex

    PubMed Central

    Vinson, Gavin P; Brennan, Caroline H

    2013-01-01

    Substantial evidence shows that the hypophyseal–pituitary–adrenal (HPA) axis and corticosteroids are involved in the process of addiction to a variety of agents, and the adrenal cortex has a key role. In general, plasma concentrations of cortisol (or corticosterone in rats or mice) increase on drug withdrawal in a manner that suggests correlation with the behavioural and symptomatic sequelae both in man and in experimental animals. Corticosteroid levels fall back to normal values in resumption of drug intake. The possible interactions between brain corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC) products and the systemic HPA, and additionally with the local CRH–POMC system in the adrenal gland itself, are complex. Nevertheless, the evidence increasingly suggests that all may be interlinked and that CRH in the brain and brain POMC products interact with the blood-borne HPA directly or indirectly. Corticosteroids themselves are known to affect mood profoundly and may themselves be addictive. Additionally, there is a heightened susceptibility for addicted subjects to relapse in conditions that are associated with change in HPA activity, such as in stress, or at different times of the day. Recent studies give compelling evidence that a significant part of the array of addictive symptoms is directly attributable to the secretory activity of the adrenal cortex and the actions of corticosteroids. Additionally, sex differences in addiction may also be attributable to adrenocortical function: in humans, males may be protected through higher secretion of DHEA (and DHEAS), and in rats, females may be more susceptible because of higher corticosterone secretion. PMID:23825159

  14. Primitive neuroectodermal adrenal gland tumour.

    PubMed

    Tsang, Y P; Lang, Brian H H; Tam, S C; Wong, K P

    2014-10-01

    Ewing's sarcoma, also called primitive neuroectodermal tumour of the adrenal gland, is extremely rare. Only a few cases have been reported in the literature. We report on a woman with adult-onset primitive neuroectodermal tumour of the adrenal gland presenting with progressive flank pain. Computed tomography confirmed an adrenal tumour with invasion of the left diaphragm and kidney. Radical surgery was performed and the pain completely resolved; histology confirmed the presence of primitive neuroectodermal tumour, for which she was given chemotherapy. The clinical presentation of this condition is non-specific, and a definitive diagnosis is based on a combination of histology, as well as immunohistochemical and cytogenic analysis. According to the literature, these tumours demonstrate rapid growth and aggressive behaviour but there are no well-established guidelines or treatment strategies. Nevertheless, surgery remains the mainstay of local disease control; curative surgery can be performed in most patients. Adjuvant chemoirradiation has been advocated yet no consensus is available. The prognosis of patients with primitive neuroectodermal tumours remains poor.

  15. Rare adrenal tumors in children.

    PubMed

    Mihai, Radu

    2014-04-01

    Apart from neuroblastomas, adrenal tumors are exceedingly rare in children and young adults. In this age group, the vast majority of patients present with clinical signs associated with excess hormone production. The most common tumor to arise from the adrenal cortex is an adrenocortical carcinoma (ACC). Similar to the situation in adults, this tumor is frequently diagnosed at a late stage and carries a very poor prognosis. ACCs require extensive/aggressive local resection followed by mitotane chemotherapy. A multidisciplinary approach is essential, and these children should be referred to units that have previous experience in managing ACCs. International registries are an invaluable source for evidence-based care, and such collaborations should be further developed in the future. Pheochromocytomas are derived from the adrenal medulla and present with symptoms caused by high secretion of catecholamines. At least one-third of these children will be found to carry genetic mutations, most commonly the RET gene (MEN2 syndrome) or the VHL gene. Open radical adrenalectomy should be offered to children with adrenocortical cancers. For all other cases, laparoscopic adrenalectomy is the treatment of choice. It is possible that the retroperitoneoscopic approach will gain increasing favor. The role of robotic adrenalectomy remains controversial.

  16. Occult adrenal insufficiency in surgical patients.

    PubMed Central

    Hubay, C A; Weckesser, E C; Levy, R P

    1975-01-01

    Eight patients admitted to a University hospital with acute surgical problems and related adrenal insufficiency were reviewed and three are presented in detail. Surgical stress and continued sepsis played major roles in the lack of responsiveness to usual modes of therapy until the adrenal insufficiency was corrected. The patients fell into three major clinical categories of adrenal insufficiency. Chronic illness and sepsis are shown to affect steroid production and metabolism, as well as adrenal responsiveness to ACTH. Pharmacologic amounts of steroids are often needed in patients with shock, gram negative sepsis and prolonged illnesses, even if normal or elevated serum cortisols are present. Therapeutic trials of cortisol administration are shown to be confusing when not accompanied by easily performed diagnostic tests of adrenal function. It is emphasized that a pretreatment serum cortisol should be obtained whenever possible. The evaluation of adrenal function is of lifelong importance to the patient. PMID:165792

  17. Laparoscopic Resection of an Adrenal Schwannoma

    PubMed Central

    Konstantinos, Toutouzas G.; Panagiotis, Kekis B.; Nikolaos, Michalopoulos V.; Ioannis, Flessas; Andreas, Manouras; Geogrios, Zografos

    2012-01-01

    Background and Objectives: Schwannomas are tumors originating from Schwann cells of the peripheral nerve sheath (neurilemma) of the neuroectoderm. Rarely, schwannomas can arise from the retroperitoneum and adrenal medulla. We describe a case of a 71-y-old woman who presented with an incidentally discovered adrenal tumor. Methods: Ultrasound and computed tomography scans revealed a lesion with solid and cystic areas originating from the left adrenal gland. The patient underwent complete laparoscopic resection of the tumor and the left adrenal gland. Results: Histopathological examination and immunohistochemical staining of the excised specimen revealed a benign schwannoma measuring 5.5×5×3.7 cm. To our knowledge, few other cases of laparoscopic resection of adrenal schwannomas have been reported. Conclusion: Because preoperative diagnosis of adrenal tumors is inconclusive, complete laparoscopic excision allows for definitive diagnosis with histological evaluation and represents the treatment of choice. PMID:23484583

  18. [Virilizing adrenal ganglioneuroma : A rare differential diagnosis in testosterone secreting adrenal tumours].

    PubMed

    Gaisa, N T; Klöppel, G; Brehmer, B; Neulen, J; Stephan, P; Knüchel, R; Donner, A

    2009-09-01

    Testosterone secreting tumours of the adrenal glands are usually adrenal carcinomas or adenomas. Here we report the rare case of an adrenal ganglioneuroma with ectopic Leydig cells, a so-called virilizing adrenal ganglioneuroma. Clinically it is characterized by symptoms of virilization, histologically by the occurrence of a population of eosinophilic cells. In the absence of crystalloids of Reinke this cell population can be identified as Leydig cells based on positive immunohistochemical staining of inhibin and calretinin.

  19. [Development of the human adrenal glands].

    PubMed

    Folligan, K; Bouvier, R; Targe, F; Morel, Y; Trouillas, J

    2005-09-01

    The human adrenal is an endocrine gland located at the superior part of the kidney. Composed of the adrenal cortex of mesoblastic origin and the adrenal medulla of neuroectoblastic origin, the human fetal adrenal grows considerably during the first three months of development. From 12 to 18 weeks of development (WD), the weight of the adrenals increases seven-fold. The gland's weight doubles from 18 to 28 WD and from 28 to 36 WD. At birth, the two adrenals weigh on average 10 g. At the 8th week, two zones are individualized in the adrenal cortex: the definitive zone and the fetal inner zone. At the second trimester, according to ultrastructural and biochemical studies, a third zone, called the transition zone, is individualized between the definitive zone and the fetal inner zone. The definitive zone persists, but the origin of the three zones (glomerular, fascicular and reticular) of adult adrenal cortex is not known. The fetal inner zone regresses from the 5th month of gestation and disappears totally one year after birth. At the 8th week, the immature neuroblasts migrate to the definitive zone, then to the fetal inner zone to compose the adrenal medulla, which develops essentially after birth and during the first year. Before the 10th week, the human fetal adrenal is able to produce steroid hormones, in particular dehydroepiandrosterone sulfate (DHEA-S); the secretion of cortisol remains discussed. The development of the human fetal adrenal is complex and is under the control of hormones (ACTH, LH and betaHCG), growth factors (ACTH essentially) and transcription factors (essentially SF1 and DAX-1). Knowledge of morphological and molecular phenomena of this development permits to understand the pathophisiology of congenital adrenal deficiencies.

  20. Metabolism of adrenal cholesterol in man

    PubMed Central

    Borkowski, Abraham; Delcroix, Claude; Levin, Sam

    1972-01-01

    The kinetics of plasma and adrenal cholesteral equilibration were analyzed in patients undergoing bilateral adrenalectomy for generalized mammary carcinoma. A biological model is proposed to help in the understanding of adrenal cholesterol physiology. It comprises two intracellular compartments: (1) A compartment of free adrenal cholesterol which is small (of the order of 17 mg) but turns over very fast; it is renewed approximately 8 times per day: 3 times by the inflow of free plasma cholesterol, and 5 times by the hydrolysis of esterified adrenal cholesterol, the contribution of adrenal cholesterol synthesis appearing to be relatively small. (2) A compartment of esterified adrenal cholesterol which is 20 times larger; it is constantly renewed by in situ esterification and hydrolysis with a daily fractional turnover rate of the order of 0.25. The direct and selective accumulation of plasma cholesteryl esters is practically absent. Only free adrenal cholesterol returns to plasma, mostly after conversion into steroid “hormones.” However small the synthesis of adrenal cholesterol may be, it seems more important in the zona “reticularis.” On the other hand, the inflow of plasma cholesterol and the turnover of the free adrenal compartment tend to be faster in the zona “fasciculata.” The equilibration of plasma and adrenal cholesterol can proceed unmodified under conditions of ACTH suppression. In one patient with Cushing's disease the size of the two adrenal compartments was clearly increased but their equilibration with plasma cholesterol proceeded normally. In another patient the kinetics of hydrocortisone corresponded to those of free adrenal cholesterol in the control studies. PMID:4338119

  1. The adrenal medulla and Parkinson's disease.

    PubMed

    Stoddard, S L

    1994-01-01

    This paper reviews the literature describing the condition of the adrenal medulla in Parkinson's disease. Parkinson's disease is a neurodegenerative disorder that is characterized primarily by the loss of dopaminergic neurons in the substantia nigra. Clinical observations have revealed that Parkinson's disease is also frequently accompanied by a variety of autonomic symptoms. The adrenal medulla is a major component of the autonomic nervous system. However, until recently this organ has not been of particular interest in Parkinson's disease. Early studies found histologic abnormalities in adrenal medullary cells, and several groups measured urinary and plasma catecholamines to determine general autonomic status. In the late 1980s adrenal medullary tissue was first transplanted to the caudate nucleus in an attempt to augment the decreased levels of dopamine, and thus treat the symptoms of Parkinson's disease. At this time the status of the adrenal medulla in this disease became clinically important. We measured the total catecholamine content of the parkinsonian adrenal medulla in tissue collected both at autopsy and in conjunction with adrenal-caudate transplants. Adrenal medullary catecholamines and several neuropeptides were severely depressed in parkinsonian glands. Thus, the adrenal medulla appears to be a target of the peripheral manifestations of Parkinson's disease.

  2. Imaging of adrenal and renal hemorrhage.

    PubMed

    Hammond, Nancy A; Lostumbo, Antonella; Adam, Sharon Z; Remer, Erick M; Nikolaidis, Paul; Yaghmai, Vahid; Berggruen, Senta M; Miller, Frank H

    2015-10-01

    Hemorrhage of the kidneys and adrenal glands has many etiologies. In the adrenal glands, trauma, anticoagulation, stress, sepsis, surgery, and neoplasms are common causes of hemorrhage. In the kidneys, reasons for hemorrhage include trauma, bleeding diathesis, vascular diseases, infection, infarction, hemorrhagic cyst rupture, the Antopol-Goldman lesion, and neoplasms. Angiomyolipoma and renal cell carcinoma are the neoplasms most commonly associated with hemorrhage in the kidneys and adrenal cortical carcinoma, metastases, and pheochromocytoma are associated with hemorrhage in the adrenal glands. Understanding the computed tomography and magnetic resonance imaging features, and causes of hemorrhage in the kidneys and adrenal glands is critical. It is also important to keep in mind that mimickers of hemorrhage exist, including lymphoma in both the kidneys and adrenal glands, and melanoma metastases in the adrenal glands. Appropriate imaging follow-up of renal and adrenal hemorrhage should occur to exclude an underlying malignancy as the cause. If there is suspicion for malignancy that cannot be definitively diagnosed on imaging, surgery or biopsy may be warranted. Angiography may be indicated when there is a suspected underlying vascular disease. Unnecessary intervention, such as nephrectomy, may be avoided in patients with benign causes or no underlying disease. Appropriate management is dependent on accurate diagnosis of the cause of renal or adrenal hemorrhage and it is incumbent upon the radiologist to determine the etiology.

  3. Unilateral adrenal enlargement due to Histoplasma capsulatum.

    PubMed

    Swartz, M A; Scofield, R H; Dickey, W D; Kirk, J L; Wilson, D A; Pitha, J V; Muchmore, H G

    1996-10-01

    Human infection with Histoplasma capsulatum runs the gamut from asymptomatic to disseminated disease. CT-directed fine-needle aspiration of bilaterally enlarged adrenal glands has been used in diagnosing serious infections with this ubiquitous organism. Three cases have previously been reported in which H. capsulatum infection caused unilateral adrenal enlargement; this enlargement was diagnosed post-mortem. We describe three patients with unilateral adrenal enlargement due to H. capsulatum whose conditions were diagnosed antemortem. We encourage clinicians to include infection with H. capsulatum as well as other granulomatous diseases and tumors in the differential diagnosis of unilateral adrenal enlargement.

  4. Adrenal scan in 17-alpha-hydroxylase deficiency: false indication of adrenal adenoma

    SciTech Connect

    Shore, R.M.; Lieberman, L.M.; Newman, T.J.; Friedman, A.; Bargman, G.J.

    1981-07-01

    A patient who was thought to have testicular feminization syndrome and primary aldosteronism had an adrenal scan that suggested an adrenal adenoma. After later diagnosis of 17-alpha-hydroxylase deficiency, she was treated with glucocorticoids rather than surgery. Her clinical course and a repeat adrenal scan confirmed she did not have a tumor.

  5. Is There Such a Thing as Adrenal Fatigue?

    MedlinePlus

    ... adrenal insufficiency caused by chronic stress. The unproven theory behind adrenal fatigue is that your adrenal glands ... feel good. Existing blood tests, according to this theory, aren't sensitive enough to detect such a ...

  6. Effect of angiotensin II, ATP, and ionophore A23187 on potassium efflux in adrenal glomerulosa cells

    SciTech Connect

    Lobo, M.V.; Marusic, E.T.

    1986-02-01

    Angiotensin II stimulus on perifused bovine adrenal glomerulosa cells elicited an increase in 86Rb efflux from cells previously equilibrated with the radioisotope. When 45Ca fluxes were measured under similar conditions, it was observed that Ca and Rb effluxes occurred within the first 30 s of the addition of the hormone and were independent of the presence of external Ca. The 86Rb efflux due to angiotensin II was inhibited by quinine and apamin. The hypothesis that the angiotensin II response is a consequence of an increase in the K permeability of the glomerulosa cell membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated 86Rb or K loss (as measured by an external K electrode). This increased K conductance was also seen with 10(-4) M ATP. Quinine and apamin greatly reduced the effect of ATP or A23187 on 86Rb or K release in adrenal glomerulosa cells. The results suggest that Ca-dependent K channels or carriers are present in the membranes of bovine adrenal glomerulosa cells and are sensitive to hormonal stimulus.

  7. Physicochemical properties of ionic and non-ionic biocompatible hydrogels in water and cell culture conditions: Relation with type of morphologies of bovine fetal fibroblasts in contact with the surfaces.

    PubMed

    Rivero, Rebeca; Alustiza, Fabrisio; Capella, Virginia; Liaudat, Cecilia; Rodriguez, Nancy; Bosch, Pablo; Barbero, Cesar; Rivarola, Claudia

    2017-07-11

    Cationic, anionic and non-ionic hydrogels having acrylamide polymer backbones were synthesized via free radical polymerization with N,N-methylenebisacrylamide (BIS) as crosslinker. The chemical structures of the hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR). Physicochemical properties such as swelling kinetic, maximum swelling capacity, volume phase transition temperature (VPTT) and wettability (static water contact angle) of hydrogels swollen in aqueous and cell culture medium, at room and cell culture temperatures were studied. In order to correlate the surface properties of the hydrogels and cellular adhesivity of bovine fetal fibroblasts (BFFs), cellular behaviour was analyzed by inverted fluorescence optical microscopy and atomic force microscopy (AFM). MTT assay demonstrated that the number of viable cells in contact with hydrogels does not significantly change in comparison to a control surface. Flattened and spindle-shaped cells and cell spheroids were the adopted morphologies during first days of culture on different hydrogels. Cell spheroids were easily obtained during the first 5days of culture in contact with PNIPAM-co-20%HMA (poly (N-isopropylacrylamide-co-20%N-acryloyl-tris-(hydroxymethyl)aminomethane)) hydrogel surface. After 15days of culture all hydrogels showed high adhesion and visual proliferation. According to obtained results, non-ionic and hydrophilic surfaces with moderated wettability induce the formation of BFFs cell spheroids. These hydrogel surfaces could be used in clinical and biochemical treatments at laboratory level to cell growth and will allow generating the base for future biotechnologic platform. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production.

    PubMed

    Jimenez-Krassel, F; Ireland, J J

    2002-01-01

    The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5 mm in diameter), medium (6 to 14 mm), or the largest (6 to 19 mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5alpha-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48 h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol > progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P < 0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells

  9. A Case Report of Bilateral Sarcomatoid Carcinoma of Adrenal Glands With Adrenal Insufficiency.

    PubMed

    Ishikawa, Noriyoshi; Nagase, Mamiko; Takami, Saki; Araki, Asuka; Ishikawa, Nahoko; Koike, Chiaki; Shiina, Hiroaki; Maruyama, Riruke

    2016-12-01

    Adrenocortical carcinomas are relatively rare, but they are considered to be highly aggressive malignant tumors. Sarcomatoid carcinomas represent an even more aggressive type. Bilateral malignant adrenal tumors are extraordinary rare, except for those that represent metastatic spread from a primary neoplasm. Here we report a case of a 69-year-old woman who presented symptoms that raised strong suspicions of adrenal insufficiency. Bilateral adrenal masses, identified in the imaging study, were responsible for the clinical manifestation and surgically resected. Surgical specimens of the bilateral adrenal tumors shared histological features compatible with sarcomatoid carcinoma. It was very difficult to confirm that the sarcomatoid carcinomas were derived from the cortex of the adrenal glands, but careful morphological observation and the panel of antibodies used for immunohistochemistry made the diagnosis possible. This is the first report of sarcomatoid carcinomas involving both adrenal glands. It should be emphasized that sarcomatoid carcinoma can arise bilaterally from even functionally impaired adrenal glands. © The Author(s) 2016.

  10. Delayed Diagnosis of Graves’ Thyrotoxicoisis Presenting as Recurrent Adrenal Crisis in Primary Adrenal Insufficiency

    PubMed Central

    Naik, Dukhabandhu; Jebasingh, K Felix

    2016-01-01

    Adrenal crisis is a potential life threatening complication. The common causes of adrenal crisis are infections, surgical stress and abrupt cessation of steroid medications. Endocrine causes like Graves’ disease with thyrotoxicosis is one of the less common causes of an adrenal crisis. We report a 42-year-old female who presented with recurrent episodes of adrenal crisis due to delayed diagnosis of thyrotoxicosis. She was initially treated with Carbimazole followed by Radio-iodine ablation and currently she is euthyroid. Her adrenal insufficiency was initially treated with hydrocortisone during the time of adrenal crisis followed by Prednisolone 5 mg once daily in the morning along with fludrocortisone 50 mcg once daily. This case highlights the need for high index of suspicion and less common causes like thyrotoxicosis should be ruled out in patients with adrenal crisis. PMID:27190873

  11. Unilateral adrenal hemorrhagic infarction in essential thrombocythemia.

    PubMed

    Burnet, G; Lambert, M; Annet, L; Lefebvre, C

    2015-12-01

    Adrenal hemorrhage is a rare disease associated with various conditions. We report a case of a 68-year-old woman with abdominal and back pain. The diagnostic work-up showed a left adrenal gland infarction associated with essential thrombocythemia. Treatment consisted in painkillers and treating the underlying condition in order to prevent further thrombotic events.

  12. Computed tomographic findings in bilateral adrenal tuberculosis

    SciTech Connect

    Wilms, G.E.; Baert, A.L.; Kint, E.J.; Pringot, J.H.; Goddeeris, P.G.

    1983-03-01

    The computed tomographic (CT) features of bilateral adrenal tuberculosis are reported in two cases that demonstrate two typical different clinical and morphological manifestations of the disease. The incidence and CT appearance of adrenal tuberculosis are discussed, with emphasis on differential diagnosis.

  13. Glucocorticoid-suppressible hyperaldosteronism and adrenal tumors occurring in a single French pedigree.

    PubMed Central

    Pascoe, L; Jeunemaitre, X; Lebrethon, M C; Curnow, K M; Gomez-Sanchez, C E; Gasc, J M; Saez, J M; Corvol, P

    1995-01-01

    Glucocorticoid-suppressible hyperaldosteronism is a dominantly inherited form of hypertension believed to be caused by the presence of a hybrid CYP11B1/CYP11B2 gene which has arisen from an unequal crossing over between the two CYP11B genes in a previous meiosis. We have studied a French pedigree with seven affected individuals in which two affected individuals also have adrenal tumors and two others have micronodular adrenal hyperplasia. One of the adrenal tumors and the surrounding adrenal tissue has been removed, giving a rare opportunity to study the regulation and action of the hybrid gene causing the disease. The hybrid CYP11B gene was demonstrated to be expressed at higher levels than either CYP11B1 or CYP11B2 in the cortex of the adrenal by RT-PCR and Northern blot analysis. In situ hybridization showed that both CYP11B1 and the hybrid gene were expressed in all three zones of the cortex. In cell culture experiments hybrid gene expression was stimulated by ACTH leading to increased production of aldosterone and the hybrid steroids characteristic of glucocorticoid-suppressible hyperaldosteronism. The genetic basis of the adrenal pathologies in this family is not known but may be related to the duplication causing the hyperaldosteronism. Images PMID:7593610

  14. Determination of catecholamines in single adrenal medullary cells by capillary electrophoresis and laser-induced native fluorescence

    SciTech Connect

    Chang, H.T.; Yeung, E.S. |

    1995-03-15

    The present study demonstrates that native fluroescence detection combined with capillary electrophoresis separation at low pH provides high sensitivity (down to nanomolar), high resolution, high speed, and low interference for the analysis of catecholamines. Further, this method has been employed successfully for the measurement of the amounts of epinephrine and norepinephrine in individual bovine adrenal medullary cells. Application of this method to the study of neurochemistry is promising. 46 refs., 7 figs., 2 tabs.

  15. ACTH-Independent Cushing’s Syndrome with Bilateral Micronodular Adrenal Hyperplasia and Ectopic Adrenocortical Adenoma

    PubMed Central

    Louiset, Estelle; Gobet, Françoise; Libé, Rossella; Horvath, Anelia; Renouf, Sylvie; Cariou, Juliette; Rothenbuhler, Anya; Bertherat, Jérôme; Clauser, Eric; Grise, Philippe; Stratakis, Constantine A.; Kuhn, Jean-Marc; Lefebvre, Hervé

    2010-01-01

    Context: Bilateral micronodular adrenal hyperplasia and ectopic adrenocortical adenoma are two rare causes of ACTH-independent Cushing’s syndrome. Objective: The aim of the study was to evaluate a 35-yr-old woman with ACTH-independent hypercortisolism associated with both micronodular adrenal hyperplasia and ectopic pararenal adrenocortical adenoma. Design and Setting: In vivo and in vitro studies were performed in a University Hospital Department and academic research laboratories. Intervention: Mutations of the PRKAR1A, PDE8B, and PDE11A genes were searched for in leukocytes and adrenocortical tissues. The ability of adrenal and adenoma tissues to synthesize cortisol was investigated by immunohistochemistry, quantitative PCR, and/or cell culture studies. Main Outcome Measure: Detection of 17α-hydroxylase and 21-hydroxylase immunoreactivities, quantification of CYP11B1 mRNA in adrenal and adenoma tissues, and measurement of cortisol levels in supernatants by radioimmunological assays were the main outcomes. Results: Histological examination of the adrenals revealed nonpigmented micronodular cortical hyperplasia associated with relative atrophy of internodular cortex. No genomic and/or somatic adrenal mutations of the PRKAR1A, PDE8B, and PDE11A genes were detected. 17α-Hydroxylase and 21-hydroxylase immunoreactivities as well as CYP11B1 mRNA were detected in adrenal and adenoma tissues. ACTH and dexamethasone activated cortisol secretion from adenoma cells. The stimulatory action of dexamethasone was mediated by a nongenomic effect involving the protein kinase A pathway. Conclusion: This case suggests that unknown molecular defects can favor both micronodular adrenal hyperplasia and ectopic adrenocortical adenoma associated with Cushing’s syndrome. PMID:19915020

  16. Extensive expertise in endocrinology. Adrenal crisis.

    PubMed

    Allolio, Bruno

    2015-03-01

    Adrenal crisis is a life-threatening emergency contributing to the excess mortality of patients with adrenal insufficiency. Studies in patients on chronic replacement therapy for adrenal insufficiency have revealed an incidence of 5-10 adrenal crises/100 patient years and suggested a mortality rate from adrenal crisis of 0.5/100 patient years. Patients with adrenal crisis typically present with profoundly impaired well-being, hypotension, nausea and vomiting, and fever responding well to parenteral hydrocortisone administration. Infections are the major precipitating causes of adrenal crisis. Lack of increased cortisol concentrations during infection enhances pro-inflammatory cytokine release and sensitivity to the toxic effects of these cytokines (e.g. tumour necrosis factor alpha). Furthermore, pro-inflammatory cytokines may impair glucocorticoid receptor function aggravating glucocorticoid deficiency. Treatment of adrenal crisis is simple and highly effective consisting of i.v. hydrocortisone (initial bolus of 100  mg followed by 200  mg over 24  h as continuous infusion) and 0.9% saline (1000  ml within the first hour). Prevention of adrenal crisis requires appropriate hydrocortisone dose adjustments to stressful medical procedures (e.g. major surgery) and other stressful events (e.g. infection). Patient education is a key for such dose adjustments but current education concepts are not sufficiently effective. Thus, improved education strategies are needed. Every patient should carry an emergency card and should be provided with an emergency kit for parenteral hydrocortisone self-administration. A hydrocortisone pen would hold a great potential to lower the current barriers to hydrocortisone self-injection. Improved patient education and measures to facilitate parenteral hydrocortisone self-administration in impending crisis are expected to significantly reduce morbidity and mortality from adrenal crisis. © 2015 European Society of Endocrinology.

  17. Synthesis of adrenal peptide E and some of its biological activities.

    PubMed

    Heimer, E P; Lambros, T J; Felix, A M; Fleminger, G; Li, C H; Westphal, M; Meienhofer, J

    1983-09-01

    A synthesis of peptide E, a highly potent, 25-amino acid adrenal opioid peptide containing both a [Met]enkephalin at the NH2-terminus and [Leu]enkephalin sequence at the COOH-terminus, originally isolated from bovine adrenal medulla [D. L. Kilpatrick, T. Taniguchi, B. N. Jones, A. S. Stern, J. E. Shively, J. Hullihan, S. Kimura, S. Stein, and S. Udenfriend (1981) Proc. Natl. Acad. Sci. USA 78, 3265-3268], is reported. The synthesis was accomplished by the solid-phase method employing the 4-(aminoacyloxymethyl)phenylacetamidomethyl(Pam)-copoly(styrene-1% divinylbenzene) resin. Two synthetic strategies (N-indole formyl protected vs unprotected tryptophan) were followed and results compared and evaluated. It was determined that peptide E prepared with protection of tryptophan (residues 13 and 14) was preferred and gave final product that was readily purified by HPLC. The biological activity of the synthetic material was found to be equivalent to the reported activity of the natural compound.

  18. Peptide F (pro-enkephalin fragment): radioimmunoassay, and stress-induced changes in adrenal

    SciTech Connect

    Alessi, N.; Taylor, L.; Akil, H.

    1982-10-18

    Utilizing a nine amino-acid (Asp-Glu-Leu-Tyr-Pro-Leu-Glu-Val-Glu) non-enkephalin containing fragment of Peptide F from the pro-enkephalin molecule, a radioimmunoassay was developed. Extraction of bovine, rat, and guinea pig adrenomedullary preparations demonstrated this fragment to be present and apparently partially conserved across species. In rats, acute inescapable foot-shock stress led to a significant decrease of the immunoreactive material in the adrenal medulla. Chronic daily stress for two weeks resulted in an inability of the adrenals to alter F levels upon subsequent stress. The existence of F-like immunoreactivity and its alteration by environmental manipulation, suggest that it may play a unique physiological role.

  19. Improvement of sensitivity for Mycobacterium avium subsp. paratuberculosis (MAP) detection in bovine fecal samples by specific duplex F57/IC real-time and conventional IS900 PCRs after solid culture enrichment.

    PubMed

    Fawzy, Ahmad; Eisenberg, Tobias; El-Sayed, Amr; Zschöck, Michael

    2015-04-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants and a probable pathogen of Crohn's disease in humans. Accurate, cost-effective, and time-relevant diagnostics are the basis for efficient control programs. This study was conducted as an attempt to re-evaluate MAP detection improvement by coupling solid media enrichment to a more specific IS900 conventional PCR and a very specific F57/IC real-time PCR. In a spiking experiment, we investigated the improvement of molecular-based MAP detection in feces after a culture-based enrichment step into Herrold's egg yolk media with mycobactin J (HEYM-MJ) for different time intervals, when compared to traditional culture. Detection limit of culture was 0.33 × 10(4) bacteria × g(-1) (33 CFU g(-1)), while that of IS900 PCR when coupled with an enrichment step for 2, 4, and 6 weeks was 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(4) (33 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Whereas the detection limits of F57/IC real-time PCR after the enrichment step for the same time intervals were 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(3) (3.3 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Altogether, enrichment of bovine fecal samples into solid media increased the sensitivity of specific molecular detection of MAP using IS900 conventional PCR and duplex F57/IC real-time PCR and offers an expedited and accurate alternative for MAP detection in bovine feces. Validation of these results is further recommended using field bovine fecal samples.

  20. mRNA expression profile of the TNF-α system in LH-induced bovine preovulatory follicles and effects of TNF-α on gene expression, ultrastructure and expansion of cumulus-oocyte complexes cultured in vitro.

    PubMed

    Silva, A W B; Bezerra, F T G; Glanzner, W G; Dos Santos, J T; Dau, A M P; Rovani, M T; Ilha, G F; Costa, J J N; Cunha, E V; Donato, M A M; Peixoto, C A; Gonçalves, P B D; Bordignon, V; Silva, J R V

    2017-03-01

    This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.

  1. Testicular Adrenal Rest Tumors in Patients with Congenital Adrenal Hyperplasia

    PubMed Central

    Şentürk Mutlu, Fatma; Eren, Erdal; Paşa, Aliye Özlem; Sağlam, Halil; Tarım, Ömer

    2012-01-01

    Objective: Early diagnosis and treatment of testicular adrenal rest tumors (TART) is important for gonadal functions and fertility protection in boys with congenital adrenal hyperplasia (CAH). In this descriptive study, we investigated the prevalence of TART in boys with 21-hydroxylase deficient (21OHD) CAH followed in our pediatric endocrine clinic. Methods: The study group consisted of 14 male patients with a mean age of 9.6±5.1 (range: 0.8-18.3) years. Six (42.9%) of the 14 patients were diagnosed as having salt-wasting type (SW) and eight (57.1%) patients - as having the simple virilizing (SV) form of 21OHD. Mean age at diagnosis was 2.9±2.7 (range: 0.03-6.3) years. Two different radiologists performed scrotal ultrasonography. Chronological age, bone age, and anthropometric measurements were evaluated. Serum adrenocorticotropic hormone (ACTH), 17-alpha-hydroxyprogesterone (17OHP) and androstenedione levels were also evaluated in all patients during the follow-up period. Results: Scrotal ultrasonography revealed bilateral TART in two patients (14.3%) and testicular microlithiasis (TM) in four patients (28.6%). One patient had both TART and TM bilaterally. During the follow-up period, the mean serum adrenocorticotropic hormone, 17OHP and androstenedione levels in the total group of patients were 130.0±179.1 pg/mL (21.7-726.5), 5.8±3.3 ng/mL (0.8-11.4) and 4.3±4.1 (0.2-11.0) ng/mL, respectively. Conclusions: Microlithiasis or TART may be frequently encountered during the follow-up of patients with CAH. In order to prevent late complications including infertility, we suggest that ultrasonographic evaluations be performed yearly in all male CAH patients. Conflict of interest:None declared. PMID:22672867

  2. Natural course of benign adrenal incidentalomas in subjects with extra-adrenal malignancy.

    PubMed

    Yener, Serkan; Ertilav, Senem; Secil, Mustafa; Akinci, Baris; Demir, Tevfik; Comlekci, Abdurrahman; Yesil, Sena

    2009-08-01

    Patients with extra-adrenal malignancies are diagnosed increasingly with benign adrenal tumors, as well as non-oncology subjects. We aimed to demonstrate the natural course of adrenal adenomas in terms of mass size and hormonal status in oncology and non-oncology subjects. We also compared the characteristics and behavior of adrenal adenomas with adrenal malignancies. In our registry of adrenal tumors (n = 335), we prospectively evaluated 29 oncology subjects (EAM+) and age, gender, and follow-up duration matched 110 non-oncology subjects (EAM-) with adrenal adenomas. Median follow-up was 24 months. We also included 16 subjects with adrenal malignancies (primary; 3 and metastasis; 13). Tumor size was followed-up with CT or MRI at 6th and 12th months and annually in subsequent visits. Hormonal assessment was repeated at the 6th month after the initial visit and annually in subsequent visits. Initial tumor size, mean increase in tumor size, and number of subjects who showed mass enlargement or developed subclinical Cushing Syndrome were comparable (P > 0.05) between EAM+ and EAM- groups. Subjects with malignant adrenal tumors were older (P = 0.06), had larger tumors at presentation (P < 0.001), and showed mass enlargement during a shorter follow-up duration (P < 0.001). Oncology subjects with adrenal adenomas featured similar baseline and follow-up parameters in terms of mass enlargement and development of subclinical Cushing Syndrome when compared with non-oncology subjects. Malignant adrenal tumors were characterized with large, rapidly growing tumors of older ages. Conservative approach can be suggested to oncology subjects for adrenal adenomas unless clinical and radiological suspicion of adrenal malignancy is present.

  3. Regulation of ACTH-induced steroidogenesis in human fetal adrenals by rTNF-alpha.

    PubMed

    Jäättelä, M; Carpén, O; Stenman, U H; Saksela, E

    1990-01-22

    The presence of tumor necrosis factor type alpha (TNF-alpha) in different fetal tissue and adult adrenal extracts was investigated by radioimmunoassay (RIA). Measurable levels of TNF-alpha were found in 12/22 fetal adrenals, but in none of the seven adult adrenals studied. Since it is known that (i) steroidogenesis in fetal adrenals differs greatly from that in adult glands by having higher androgen/corticosteroid ratio, (ii) and that macrophage-derived factors may cause adrenocortical suppression, the effect of TNF-alpha on corticotropin-induced steroidogenesis in primary cultures of human fetal adrenals was studied. Results show that TNF-alpha effectively suppresses the production of cortisol and shifts the steroid synthesis towards androgen production. The effect was not accompanied by any change in cell viability and could be neutralized by addition of polyclonal rabbit anti-TNF-alpha antiserum to cell cultures. These results suggest that TNF-alpha may take part in the regulation of human fetal steroidogenesis within the network of the fetoplacental unit via inhibition of the cortisol synthesis.

  4. Corticomedullary mixed tumor of the adrenal gland.

    PubMed

    Wieneke, J A; Thompson, L D; Heffess, C S

    2001-10-01

    Corticomedullary mixed tumors of the adrenal gland are quite rare, with only five well-documented cases reported in the literature.(1-4) Herein, we report the light microscopic and immunohistochemical features of two cases of this rare tumor. Patient 1 is a 34-year-old woman who presented with hypertension, hair loss, and amenorrhea of 1-year duration. Patient 2 is a 52-year-old woman who presented with flank pain and what appeared to be a renal mass on arteriogram with no history of hypertension, Cushing's syndrome, or other endocrine abnormalities. At surgery, the tumor was noted to arise from the adrenal gland rather than the kidney and adrenalectomy was performed. In both cases, the surgically resected specimens consisted of a well-circumscribed, single adrenal mass surrounded by a rim of uninvolved adrenal cortical tissue. The tumors were composed of adrenal cortical cells intimately admixed with pheochromocytes. Immunohistochemical studies highlighted these two cellular components. The pheochromocytes were strongly reactive with chromogranin and the sustentacular cells with S-100 protein, whereas the adrenal cortical cells reacted specifically with inhibin. Thus, we report two additional cases of mixed corticomedullary tumor of the adrenal gland. Ann Diagn Pathol 5:304-308, 2001. This is a US government work. There are no restrictions on its use.

  5. Clinicopathological correlates of adrenal Cushing's syndrome.

    PubMed

    Duan, Kai; Gomez Hernandez, Karen; Mete, Ozgur

    2015-03-01

    Endogenous Cushing's syndrome is a rare endocrine disorder that incurs significant cardiovascular morbidity and mortality, due to glucocorticoid excess. It comprises adrenal (20%) and non-adrenal (80%) aetiologies. While the majority of cases are attributed to pituitary or ectopic corticotropin (ACTH) overproduction, primary cortisol-producing adrenal cortical lesions are increasingly recognised in the pathophysiology of Cushing's syndrome. Our understanding of this disease has progressed substantially over the past decade. Recently, important mechanisms underlying the pathogenesis of adrenal hypercortisolism have been elucidated with the discovery of mutations in cyclic AMP signalling (PRKACA, PRKAR1A, GNAS, PDE11A, PDE8B), armadillo repeat containing 5 gene (ARMC5) a putative tumour suppressor gene, aberrant G-protein-coupled receptors, and intra-adrenal secretion of ACTH. Accurate subtyping of Cushing's syndrome is crucial for treatment decision-making and requires a complete integration of clinical, biochemical, imaging and pathology findings. Pathological correlates in the adrenal glands include hyperplasia, adenoma and carcinoma. While the most common presentation is diffuse adrenocortical hyperplasia secondary to excess ACTH production, this entity is usually treated with pituitary or ectopic tumour resection. Therefore, when confronted with adrenalectomy specimens in the setting of Cushing's syndrome, surgical pathologists are most commonly exposed to adrenocortical adenomas, carcinomas and primary macronodular or micronodular hyperplasia. This review provides an update on the rapidly evolving knowledge of adrenal Cushing's syndrome and discusses the clinicopathological correlations of this important disease.

  6. Sonography of the adrenal glands in the adult.

    PubMed

    Kim, Kyoung Won; Kim, Jeong Kon; Choi, Hyuck Jae; Kim, Mi-hyun; Lee, Jeongjin; Cho, Kyoung-Sik

    2012-01-01

    Although its capability has been overlooked, sonography can be a useful screening tool for adrenal lesion in adults. In this article, we discuss scan technique, patient positioning, and anatomic consideration for adrenal sonography in adults and illustrate sonographic appearance of normal adrenal gland as well as adrenal tumors and tumor-like lesions. Copyright © 2012 Wiley Periodicals, Inc.

  7. 9 CFR 113.309 - Bovine Parainfluenza3 Vaccine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Live Virus Vaccines § 113.309 Bovine Parainfluenza3 Vaccine. Bovine Parainfluenza3 Vaccine shall be produced from virus-bearing cell culture fluids. Only Master Seed Virus which has been established as pure, safe...

  8. Severe hyponatremia caused by hypothalamic adrenal insufficiency.

    PubMed

    Shibata, T; Oeda, T; Saito, Y

    1999-05-01

    A 60-year-old woman was admitted with severe hyponatremia. Basal values of adrenocorticotropic hormone (ACTH), thyroid hormone and cortisol were normal on admission. Impairment of water diuresis was observed by water loading test. Initially, we diagnosed her condition as the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). By provocation test, we finally confirmed that the hyponatremia was caused by hypothalamic adrenal insufficiency. The basal values of ACTH and cortisol might not be sufficient to exclude the possibility of adrenal insufficiency. Therefore, it is necessary to evaluate adrenal function by provocation test or to re-evaluate it after recovery from hyponatremia.

  9. Spontaneous Unilateral Adrenal Hemorrhage in Pregnancy

    PubMed Central

    Ebrahem, Rawaa; Munguti, Cyrus; Mortada, Rami

    2017-01-01

    Spontaneous adrenal hemorrhage (SAH) is a serious medical condition associated with variable clinical presentation depending on the extent of the hemorrhage. Pregnancy-induced adrenal hemorrhage is poorly understood. A low cortisol level in the peripartum period with radiological findings is sufficient to establish the diagnosis. Prompt hormone replacement and supportive care to ensure good clinical outcomes is crucial. Due to the potentially life-threatening complications, physicians should have a high suspicion for adrenal hemorrhage when they evaluate patients with hypotension, fatigue, and abdominal pain during the peripartum period. PMID:28191381

  10. Coexistence of Cushing syndrome from functional adrenal adenoma and Addison disease from immune-mediated adrenalitis.

    PubMed

    Colucci, Randall; Jimenez, Rafael E; Farrar, William; Malgor, Ramiro; Kohn, Leonard; Schwartz, Frank L

    2012-06-01

    A 56-year-old woman presented with an incidental adrenal adenoma and physical examination findings that included moderate obesity, a slight cervicothoracic fat pad ("buffalo hump"), increased supraclavicular fat pads, and white abdominal striae. Biochemical workup revealed elevated levels of 24-hour urinary free cortisol but normal serum morning cortisol and suppressed levels of corticotropin, suggestive of adrenal-dependent Cushing syndrome. The resected adrenal gland revealed macronodular cortical hyperplasia with a dominant nodule. Other findings included an absent cortisol response to corticotropin stimulation, presence of serum anti-21-hydroxylase antibodies, and mononuclear cell infiltration--consistent with adrenalitis. The findings represent, to the authors' knowledge, the first known case of a patient with coexistent functional cortisol-secreting macronodular adrenal tumor resulting in Cushing syndrome and immune-mediated adrenalitis resulting in Addison disease.

  11. Adrenal Collision Tumor: Coexistence of Pigmented Adrenal Cortical Oncocytoma and Ganglioneuroma

    PubMed Central

    Kim, Chungyeul

    2016-01-01

    Background. Adrenal collision tumors (ACTs), in which distinct tumors coexist without intermingling in the same adrenal gland, are rare and their actual prevalence is unknown. ACTs commonly consist of adrenal cortical adenoma, pheochromocytoma, or metastatic malignant tumor. Case Report. A 32-year-old woman who had been experiencing gastric discomfort for one month was referred to our hospital with abnormal imaging findings. The physical examination and the laboratory data including endocrine studies were unremarkable. Abdomen computed tomography (CT) and magnetic resonance imaging (MRI) showed two adjacent masses in the left suprarenal fossa, and a laparoscopic left adrenalectomy was done. Histological and immunohistochemical (IHC) examinations revealed two distinct tumors: a pigmented adrenal cortical oncocytoma (ACO) and a ganglioneuroma, respectively. Conclusion. Both tumors are rare in the adrenal gland and exist as ACTs only exceptionally rarely. This is the first reported case of coexisting oncocytoma and ganglioneuroma in the same adrenal gland to our knowledge. PMID:28053800

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

    USDA-ARS?s Scientific Manuscript database

    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...

  13. A case of adrenal Cushing’s syndrome with bilateral adrenal masses

    PubMed Central

    Guo, Ya-Wun; Hwu, Chii-Min; Won, Justin Ging-Shing; Chu, Chia-Huei

    2016-01-01

    Summary A functional lesion in corticotrophin (ACTH)-independent Cushing’s syndrome is difficult to distinguish from lesions of bilateral adrenal masses. Methods for distinguishing these lesions include adrenal venous sampling and 131I-6β-iodomethyl-19-norcholesterol (131I-NP-59) scintigraphy. We present a case of a 29-year-old Han Chinese female patient with a history of hypercholesterolaemia and polycystic ovary syndrome. She presented with a 6month history of an 8kg body weight gain and gradual rounding of the face. Serial examinations revealed loss of circadian rhythm of cortisol, elevated urinary free-cortisol level and undetectable ACTH level (<5pg/mL). No suppression was observed in both the low- and high-dose dexamethasone suppression tests. Adrenal computed tomography revealed bilateral adrenal masses. Adrenal venous sampling was performed, and the right-to-left lateralisation ratio was 14.29. The finding from adrenal scintigraphy with NP-59 was consistent with right adrenal adenoma. The patient underwent laparoscopic right adrenalectomy, and the pathology report showed adrenocortical adenoma. Her postoperative cortisol level was 3.2μg/dL, and her Cushingoid appearance improved. In sum, both adrenal venous sampling and 131I-NP-59 scintigraphy are good diagnostic methods for Cushing’s syndrome presenting with bilateral adrenal masses. Learning points The clinical presentation of Cushing’ syndrome includes symptoms and signs of fat redistribution and protein-wasting features. The diagnosis of patients with ACTH-independent Cushing’s syndrome with bilateral adrenal masses is challenging for localisation of the lesion. Both adrenal venous sampling and 131I-NP-59 scintigraphy are good methods to use in these patients with Cushing’s syndrome presenting with bilateral adrenal masses. PMID:27252858

  14. Presence of kisspeptin-like immunoreactivity in human adrenal glands and adrenal tumors.

    PubMed

    Takahashi, Kazuhiro; Shoji, Itaru; Shibasaki, Akiko; Kato, Ichiro; Hiraishi, Keisuke; Yamamoto, Hajime; Kaneko, Kiriko; Murakami, Osamu; Morimoto, Ryo; Satoh, Fumitoshi; Ito, Sadayoshi; Totsune, Kazuhito

    2010-05-01

    Kisspeptins are neuropeptides which activate the hypothalamo-pituitary gonadal axis and are considered to play important physiological roles in the reproduction. Kisspeptins have also been reported to stimulate the aldosterone secretion from the adrenal cortex. However, the expression of kisspeptins in human adrenal glands and adrenal tumors has not been clarified yet. We, therefore, studied the presence of kisspeptin-like immunoreactivity (LI) in human adrenal glands and adrenal tumors (adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas) by radioimmunoassay and immunocytochemistry. Kisspeptin-LI was detected in all the tissues examined; normal portions of adrenal glands (3.0 +/- 2.3 pmol/g wet weight, n = 21, mean +/- SD), aldosterone-producing adenomas (4.6 +/- 3.3 pmol/g wet weight, n = 10), cortisol-producing adenomas (2.7 +/- 1.4 pmol/g wet weight, n = 14), adrenocortical carcinomas (1.7 +/- 0.2 pmol/g wet weight, n = 4), and pheochromocytomas (1.8 +/- 0.8 pmol/g wet weight, n = 6). There was no significant difference in kisspeptin-LI levels among them. Immunocytochemistry showed positive kisspeptin-immunostaining in normal adrenal glands, with stronger immunostaining found in the medulla. Furthermore, positive kisspeptin-immunostaining was found in all types of adrenal tumors examined; adrenocortical adenomas, adrenocortical carcinomas, and pheochromocytomas. The intensity of kisspeptin-immunostaining in these adrenal tumors was, however, not so strong as that in normal adrenal medulla. The present study has shown for the first time the presence of kisspeptin-LI in adrenal glands and adrenal tumors.

  15. Non-Hodgkin's lymphoma involving a femur bone and bilateral adrenal glands alone with adrenal insufficiency.

    PubMed

    Iwahara, Yoshihito; Shinohara, Tsutomu; Naruse, Keishi; Komatsu, Yukihisa

    2017-01-31

    Primary bone lymphoma and primary adrenal lymphoma are rare clinicopathological entities of non-Hodgkin's lymphoma (NHL). We present the first case of diffuse large B-cell lymphoma with the involvement of a single bone and both adrenal glands alone with adrenal insufficiency. As primary extranodal NHL may have other unusual extranodal lesions, which may present unexplained clinical findings, patients with primary extranodal NHL require careful systemic examination, even when lymphadenopathy is absent. 2017 BMJ Publishing Group Ltd.

  16. Impact of increased oxygen delivery via bovine red blood cell supplementation of culturing media on select metabolic and synthetic functions of C3A hepatocytes maintained within a hollow fiber bioreactor.

    PubMed

    Gordon, Jason; Palmer, Andre F

    2005-01-01

    Hepatocytes are highly dependent upon appropriate oxygen provision for activity and viability. However, oxygen delivery to hepatocytes cultured within a hollow fiber bioreactor is believed to be problematic because of large diffusion distances, a high hepatocyte oxygen consumption rate and low aqueous media oxygen solubility. Supplementation of bioreactor media with bovine red blood cells (bRBCs) is one means of improving oxygen delivery to hepatocytes as hemoglobin contained within bRBCs binds oxygen. The impact of supplementing hepatocyte culturing media with bRBCs (approximately 5 x 10(8) bRBCs/ml) on hepatocyte activity (albumin and lactate production and glucose consumption) was studied. Decreased hepatocyte lactate production to glucose consumption ratios were found for the case when bRBCs were added to circulating culturing media, which indicated the presence of a more aerobic environment in comparison to the control (no bRBC supplementation). Additionally, albumin synthesis was found to be improved when the circulating media was supplemented with bRBCs. Our results thus support the use of bRBCs to improve oxygen delivery to hepatocytes maintained within a hollow fiber bioreactor.

  17. Radiology of the adrenals with sonography and CT

    SciTech Connect

    Mitty, H.A.; Yeh, H.C.

    1982-01-01

    The basic science and application of clinical adrenal imaging is presented. The initial chapters deal with anatomic review and methods of adrenal imaging. The bulk of the book consists of individual chapters describing pathologic entities and syndromes of adrenal disease. The final chapter deals with differentiation of adrenal lesions from masses arising in adjacent organs. There is no other single source available which so concisely presents adrenal imaging. (KRM)

  18. [Neonatal adrenal hemorrhage revealed by jaundice: a case report].

    PubMed

    Oulmaati, A; Hays, S; Mory-Thomas, N; Bretones, P; Bensaid, M; Jordan, I; Bonfils, M; Godbert, I; Picaud, J-C

    2012-04-01

    The clinical presentation of adrenal hemorrhage varies, depending on the extent of hemorrhage as well as the amount of adrenal cortex involved by the hemorrhage. We report here a case of neonatal adrenal hemorrhage revealed by late onset of neonatal jaundice. This adrenal hemorrhage most probably resulted from shoulder dystocia. The aim of this work was to focus on the fact that jaundice can be caused by adrenal hemorrhage and to emphasize the crucial importance of abdominal ultrasound in cases of persistent jaundice.

  19. Expression of the IGF and the aromatase/estrogen receptor systems in human adrenal tissues from early infancy to late puberty: implications for the development of adrenarche.

    PubMed

    Belgorosky, Alicia; Baquedano, María Sonia; Guercio, Gabriela; Rivarola, Marco A

    2009-03-01

    Adrenarche is a process of postnatal sexual maturation occurring in higher primates, in which there is an increase in the secretion of adrenal androgens. It is the consequence of a process of postnatal organogenesis characterized by the development of a new zone in the adrenal cortex, the zona reticularis (ZR). The mechanism of this phenomenon remains poorly understood, suggesting that it might be a multifactorial event. A relationship between circulating IGF-I, insulin sensitivity, and adrenal androgens has been postulated. Boys and girls have different patterns of changes in insulin sensitivity at puberty, perhaps secondary to differences in the estrogen milieu. Estrogen effects may also play a role in premature adrenarche. Peripheral or local IGF-1 actions could regulate adrenal progenitor cell proliferation and migration. Since adrenal progenitor cells as well as IGF-I and the IGF-R1 are located in the outer zone of the adrenal cortex during childhood and adolescence, this peripheral cell layer, below the capsule, may contain undifferentiated progenitor cells. Therefore, the IGF-R1 signaling pathway might positively modulate the proliferation and migration of adrenal progenitor cell to stimulate the development of adrenal zones, including ZR. However, no evidence of a direct action of IGF-I on ZR was found. In addition, a role for estrogens in the ontogenesis of ZR is suggested by the presence of aromatase (CYP19) in the subcapsular zona glomerulosa and in the adrenal medulla. Estrogens produced locally could act on ZR by interacting with estrogen receptor beta (ERbeta), but not alpha, and membrane estrogen receptor GPR-30. An estradiol-induced increase in DHEA/cortisol ratio was indeed seen in cultures of adrenocortical cells from post-adrenarche adrenals. In summary, several lines of evidence point to the action of multiple factors, such as local adrenal maturational changes and peripheral metabolic signals, on postnatal human adrenal gland ZR formation.

  20. alpha1H T-type Ca2+ channel is the predominant subtype expressed in bovine and rat zona glomerulosa.

    PubMed

    Schrier, A D; Wang, H; Talley, E M; Perez-Reyes, E; Barrett, P Q

    2001-02-01

    The low voltage-activated (T-type) Ca2+ channel has been implicated in the regulation of aldosterone secretion from the adrenal zona glomerulosa by extracellular K+ levels, angiotensin II, and ACTH. However, the identity of the specific subtype mediating this regulation has not been determined. We utilized in situ hybridization to examine the distribution of three newly cloned members of the T-type Ca2+ channel family, alpha1G, alpha1H, and alpha1I, in the rat and bovine adrenal gland. Substantial expression of only the mRNA transcript for the alpha1H-subunit was detected in the zona glomerulosa of both rat and bovine. A much weaker expression signal was detected for the alpha1H transcript in the zona fasciculata of bovine. Whole cell recordings of isolated bovine adrenal zona glomerulosa cells showed the native low voltage-activated current to be inhibited by NiCl2 with an IC50 of 6.4 +/- 0.2 microM. Because the alpha1H subtype exhibits similar NiCl2 sensitivity, we propose that the alpha1H subtype is the predominant T-type Ca2+ channel present in the adrenal zona glomerulosa.

  1. Abnormal fibrillin metabolism in bovine Marfan syndrome.

    PubMed Central

    Potter, K. A.; Hoffman, Y.; Sakai, L. Y.; Byers, P. H.; Besser, T. E.; Milewicz, D. M.

    1993-01-01

    Bovine Marfan syndrome is a disorder that closely resembles human Marfan syndrome in its clinical signs and pathological lesions. The similarities between the human and bovine diseases suggest that similar metabolic defects could be responsible. Although indirect immunofluorescent assays for fibrillin in skin biopsies did not distinguish affected cattle from control animals, cultures of skin fibroblasts of affected animals were distinguished from normal, unrelated control animals and normal half-siblings on the basis of fibrillin staining. After 72 to 96 hours in culture, stained with anti-fibrillin monoclonal antibody 201, hyperconfluent fibroblast cultures of affected cattle had less immunoreactive fibrillin than control cultures, and the staining pattern was granular rather than fibrillar. Under similar culture conditions, normal bovine aortic smooth muscle cells produced large amounts of immunoreactive fibrillin, but smooth muscle cells from a single affected cow showed markedly less fibrillin staining. In pulse-chase metabolic labeling experiments with [35S]cysteine, dermal fibroblasts from 6 affected calves, incorporated far less fibrillin into the extracellular matrix than control cells. These findings are similar to those reported in human Marfan syndrome, and they suggest that the bovine Marfan syndrome, like the human disorder, is caused by a mutation in fibrillin, leading to defective microfibrillar synthesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8456941

  2. Selective blockade of nicotinic acetylcholine receptors by pimobendan, a drug for the treatment of heart failure: reduction of catecholamine secretion and synthesis in adrenal medullary cells.

    PubMed

    Toyohira, Yumiko; Kubo, Tatsuhiko; Watanabe, Miyabi; Uezono, Yasuhito; Ueno, Susumu; Shinkai, Koji; Tsutsui, Masato; Izumi, Futoshi; Yanagihara, Nobuyuki

    2005-02-01

    Pimobendan, a Ca(2+) sensitizer, is used clinically in the treatment of chronic heart failure. Although chronic heart failure is associated with activation of the sympathetic nervous system, it remains unknown whether pimobendan affects the function of sympathetic neurons and the adrenal medulla. Here, we report the inhibitory effects of pimobendan on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Pimobendan decreased the catecholamine secretion (IC(50)=29.5 microM) elicited by carbachol, an agonist at nicotinic acetylcholine receptors, but not that elicited by veratridine, an activator of voltage-dependent Na(+) channels, or by high K(+), an activator of voltage-dependent Ca(2+) channels. Pimobendan also inhibited carbachol-induced influx of (22)Na(+) (IC(50)=25.9 microM) and (45)Ca(2+) (IC(50)=26.0 microM), but not veratridine-induced (22)Na(+) influx or high K(+)-induced (45)Ca(2+) influx. The reduction of catecholamine secretion caused by pimobendan was not overcome by increasing the concentration of carbachol. UD-CG 212, an active metabolite of pimobendan, lowered carbachol-induced catecholamine secretion with a concentration/inhibition curve similar to that of pimobendan. In experiments in situ, pimobendan suppressed both basal and carbachol-stimulated (14)C-catecholamine synthesis (IC(50)=5.3 and 4.9 microM) from [(14)C] tyrosine [but not from L: -3, 4-dihydroxyphenyl [3-(14)C] alanine ([(14)C]DOPA)], as well as tyrosine hydroxylase activity (IC(50)=3.8 and 4.3 microM). These findings suggest that pimobendan inhibits carbachol-induced catecholamines secretion and synthesis through suppression of nicotinic acetylcholine receptors.

  3. Image-Guided Ablation of Adrenal Lesions

    PubMed Central

    Yamakado, Koichiro

    2014-01-01

    Although laparoscopic adrenalectomy has remained the standard of care for the treatment for adrenal tumors, percutaneous image-guided ablation therapy, such as chemical ablation, radiofrequency ablation, cryoablation, and microwave ablation, has been shown to be clinically useful in many nonsurgical candidates. Ablation therapy has been used to treat both functioning adenomas and malignant tumors, including primary adrenal carcinoma and metastasis. For patients with functioning adenomas, biochemical and symptomatic improvement is achieved in 96 to 100% after ablation; for patients with malignant adrenal neoplasms, however, the survival benefit from ablation therapy remains unclear, though good initial results have been reported. This article outlines the current role of ablation therapy for adrenal lesions, as well as identifying some of the technical considerations for this procedure. PMID:25049444

  4. Advanced glycosylation end products in adrenal lipofuscin.

    PubMed

    Shimokawa, I; Higami, Y; Horiuchi, S; Iwasaki, M; Ikeda, T

    1998-01-01

    The present study examined the presence of advanced glycosylation end products (AGEs) in lipofuscin present in the brain and adrenal gland of aging rats by immunohistochemistry using antibodies raised against AGEs. Lipofuscin identified as yellow to brown granules emitting bright yellow to orange autofluorescence with ultraviolet light were detected in cortical neurons, cerebellar Purkinje cells, and adrenal cells in the inner part of the zona reticularis. However, none of the antibodies visualized lipofuscin in these areas. The outer part of the zona reticularis contained yellow granules emitting a faint orange autofluorescence. These granules were immunostained by an antibody that reacted with AGEs structures unrelated to the carboxymethyllysine moiety. Newly formed adrenal cortical cells are thought to migrate from the outer layer to the inner layer of the zona reticularis. Therefore, our results suggest that glycosylation-related processes are involved in lipofuscinogenesis, at least in its early stage, in the adrenal zona reticularis.

  5. Genetics Home Reference: primary macronodular adrenal hyperplasia

    MedlinePlus

    ... produced from the GNAS gene helps stimulate the activity of an enzyme called adenylate cyclase. This enzyme is involved in controlling the production of several hormones that help regulate the activity of certain endocrine glands, including the adrenal glands. ...

  6. Ancient history of congenital adrenal hyperplasia.

    PubMed

    New, Maria I

    2011-01-01

    Although there are many erudite reports on the history of endocrinology and endocrine disorders, the history of congenital adrenal hyperplasia has not been published. I have tried to review ancient as well as modern history of CAH.

  7. Adipogenesis of bovine perimuscular preadipocytes

    SciTech Connect

    Taniguchi, Masaaki; Le Luo Guan; Zhang Bing; Dodson, Michael V.; Okine, Erasmus; Moore, Stephen S.

    2008-02-01

    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-{gamma}), 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-{gamma} and SREBP-1 cooperatively play a key role to regulate the re-differentiation of bovine adipofibroblasts, during early conversion stages in vitro.

  8. The effect of bedrest on adrenal function

    NASA Technical Reports Server (NTRS)

    Leach, C. S.; Hulley, S. B.; Rambaut, P. C.; Dietlein, L. F.

    1973-01-01

    Eight male subjects were subjected to continuous bedrest for 24-80 weeks for the purpose of studying metabolic responses. Three of the subjects did supine exercises daily during part of the study. Adrenal function was examined in relation to adrenal cortical and medullary excretions. The results reveal an increase in hydrocortisone throughout the test period, a decrease in norepinephrine and no change in epinephrine. These data suggest that exercise could decrease the severity of deconditioning caused by bedrest.

  9. Primary hydatid cyst in the adrenal gland.

    PubMed

    Mohammadi, Afshin; Ghasemi-Rad, Mohammad; Oklu, Rahmi

    2014-10-23

    An elderly man presented with a 2-year history of refractory hypertension. His medical history, physical examination and laboratory findings were unremarkable. On subsequent ultrasound study for the evaluation of renal artery stenosis, a large mass obliterating the adrenal gland containing internal cystic structures was identified. A CT study confirmed the diagnosis of primary adrenal gland hydatid cyst. Following surgical resection, the patient's hypertension resolved and medications to control blood pressure were discontinued.

  10. Metabolism of adrenal cholesterol in man

    PubMed Central

    Borkowski, Abraham; Delcroix, Claude; Levin, Sam

    1972-01-01

    The synthesis of adrenal cholesterol, its esterification and the synthesis of the glucocorticosteroid hormones were studied in vitro on human adrenal tissue. It was found that the synthesis of adrenal cholesterol may normally be small in the zona “fasciculata,” particularly when compared with the synthesis of the glucocorticosteroid hormones, that it is several times higher in the zona “reticularis” where esterified cholesterol is less abundant, and that under ACTH stimulation it increases strikingly and proportionally to the degree of esterified adrenal cholesterol depletion. On the other hand, the relative rate of esterification as well as the concentration of free adrenal cholesterol are remarkably stable: they do not differ according to the adrenal zonation and are unaffected by ACTH. Furthermore, from a qualitative point of view, the relative proportions of Δ1 and Δ2 cholesteryl esters formed in situ are similar to those anticipated from their relative concentrations, suggesting that the characteristic fatty acid distribution of the adrenal cholesteryl esters results from an in situ esterification rather than from a selective uptake of the plasma cholesteryl esters. Besides, the in vitro esterification reveals a propensity to the formation of the most unsaturated cholesteryl esters. Regarding hydrocortisone and corticosterone, their synthesis tends to be more elevated in the zona “fasciculata.” Despite its higher cholesterol concentration the zona “fasciculata” should not therefore be viewed as a quiescent functional complement to the zona “reticularis” and the cortical distribution of glucocorticosteroid hormone synthesis is quite distinct from that of adrenal cholesterol synthesis. PMID:4338120

  11. Coexistence of pheochromocytoma, adrenal adenoma and hypokalemia.

    PubMed Central

    Wilkins, G. E.; Schmidt, N.; Lee-Son, L.

    1977-01-01

    A 56-year-old woman had a 22-year history of hypertension. Investigation showed hypokalemia and kaliuresis without pronounced suppression of plasma renin activity or elevation of urinary aldosterone excretion. There was biochemical evidence of catecholamine metabolite excess but the usual clinical features of pheochromocytoma were absent. Laparotomy revealed a pheochromocytoma and adrenal adenoma in the right adrenal gland. Excision of the tumours was followed by resolution of the hypertension and metabolic abnormalities. Images FIG. 2 FIG. 3 PMID:844017

  12. Testing the biocompatibility of a glutathione-containing intra-ocular irrigation solution by using an isolated perfused bovine retina organ culture model - an alternative to animal testing.

    PubMed

    Januschowski, Kai; Zhour, Ahmad; Lee, Albert; Maddani, Ramin; Mueller, Sebastien; Spitzer, Martin S; Schnichels, Sven; Schultheiss, Maximilian; Doycheva, Deshka; Bartz-Schmidt, Karl-Ulrich; Szurman, Peter

    2012-03-01

    The effects of a glutathione-containing intra-ocular irrigation solution, BSS Plus©, on retinal function and on the survival of ganglion cells in whole-mount retinal explants were studied. Evidence is provided that the perfused ex vivo bovine retina can serve as an alternative to in vivo animal testing. Isolated bovine retinas were prepared and perfused with an oxygen-saturated standard irrigation solution, and an electroretinogram was recorded to assess retinal function. After stable b-waves were detected, the isolated retinas were perfused with BSS Plus for 45 minutes. To investigate the effects of BSS Plus on photoreceptor function, 1mM aspartate was added to the irrigation solution in order to obtain a-waves, and the ERG trace was monitored for 75 minutes. For histological analysis, isolated whole retinal mounts were stored for 24 hours at 4°C, in the dark. The percentages of cell death in the retinal ganglion cell layer and in the outer and inner nuclear layers were estimated by using an ethidium homodimer-1 stain and the TUNEL assay. General swelling of the retina was examined with high-resolution optical coherence tomography. During perfusion with BSS Plus, no significant changes in a-wave and b-wave amplitudes were recorded. Retinas stored for 24 hours in BSS Plus showed a statistically significant smaller percentage (52.6%, standard deviation [SD] = 16.1%) of cell death in the retinal ganglion cell layer compared to the control group (69.6%, SD = 3.9, p = 0.0031). BSS Plus did not seem to affect short-term retinal function, and had a beneficial effect on the survival of retinal ganglion cells. This method for analysing the isolated perfused retina represents a valuable alternative for testing substances for their retinal biocompatibility and toxicity. 2012 FRAME.

  13. Black adrenal adenoma causing preclinical Cushing's syndrome.

    PubMed

    Inomoto, Chie; Sato, Haruhiro; Kanai, Genta; Hirukawa, Takashi; Shoji, Sunao; Terachi, Toshiro; Kajiwara, Hiroshi; Osamura, Robert Yoshiyuki

    2010-07-20

    Functioning black adrenal adenoma (BAA) rarely causes preclinical Cushing's syndrome (CS). In the present case, a 46-year-old Japanese Peruvian woman presented with left flank pain and hypertension. Abdominal computed tomography showed that she had a 15-mm in diameter, round, left adrenal adenoma. She had no physical features of CS, such as moon face, buffalo hump, truncal obesity, or purple striae. Endocrinological examination showed that the plasma adrenocorticotropic hormone (ACTH) level was below the detectable level, despite a serum cortisol level within the normal range. A normal cortisol circadian rhythm was not present. Dexamethasone (1 mg and 8 mg) suppression testing did not decrease serum cortisol levels to the reference levels. These findings were compatible with preclinical CS. The left adrenal adenoma was laparoscopically removed. Examination of the surgical specimen revealed unilateral double adrenal adenomas of the left adrenal gland, one of which was a BAA. The BAA measured 20 × 11 × 10 mm. Microscopically, the BAA showed proliferation of compact cells containing numerous brown-pigmented granules. There were also foci of myelolipomatous degenerative changes in the tumor. The compact cell zones remained in the adrenal cortex adjacent to the BAA showed atrophic change. These findings indicated that BAA appeared to have caused preclinical CS in this patient.

  14. [Adrenal gland insufficiency secondary to paracoccidioidomycosis].

    PubMed

    Oñate, José M; Tobón, Angela María; Restrepo, Angela

    2002-09-01

    Paracoccidioidomycosis is regularly associated with adrenal insufficiency in 10-15% of symptomatic cases, and in some instances, diagnosis of the mycosis precedes the adrenal manifestation. To establish the frequency of this association, records were reviewed of 207 cases diagnosed with mycosis at the Mycology Service of the Corporación para Investigaciones Biológicas. Six cases (2.9%) were found to have adrenal insufficiency. Patients were all males with a mean age of 67.2 years (range 48-75) and most worked in agriculture. The duration of the symptoms of adrenal damage was 4.1 months (range 2-6). All patients experienced weight loss and malaise; all had abnormal lung X-rays. Major clinical improvement was recorded after initiation of the specific treatments consisting of itraconazole, prednisolone and fluorcortisone. Diminished antibody titers against Paracoccidioides brasiliensis were also recorded after treatment. Prompt treatment re-established adrenal function and effected recovery of normal gland morphology. Consequently, early detection of hypoadrenalism in patients living in the endemic areas is necessary to avoid further adrenal damage and permits a shorter hormonal treatment period in patients afflicted by the mycosis.

  15. Effect of the microenvironment and embryo density on developmental characteristics and gene expression profile of bovine preimplantative embryos cultured in vitro.

    PubMed

    Hoelker, Michael; Rings, Franka; Lund, Qamaruddin; Ghanem, Nasser; Phatsara, Chirawath; Griese, Josef; Schellander, Karl; Tesfaye, Dawit

    2009-03-01

    The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos.

  16. Expression of adrenomedullin 2/intermedin in human adrenal tumors and attached non-neoplastic adrenal tissues.

    PubMed

    Morimoto, Ryo; Satoh, Fumitoshi; Murakami, Osamu; Hirose, Takuo; Totsune, Kazuhito; Imai, Yutaka; Arai, Yoichi; Suzuki, Takashi; Sasano, Hironobu; Ito, Sadayoshi; Takahashi, Kazuhiro

    2008-07-01

    Adrenomedullin 2/intermedin (AM2/IMD) is a new member of calcitonin/calcitonin gene-related peptide family. AM is expressed in various tumors including adrenocortical tumors and modulates tumor growth. The AM2/IMD expression has not been studied, however, in adrenal tumors. The expression of AM2/IMD and AM was therefore studied in human adrenal tumors and attached non-neoplastic adrenal tissues by immunocytochemistry (ICC). Immunoreactive (IR)-AM2/IMD was measured by RIA. Furthermore, the expression of AM2/IMD and its receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity-modifying proteins (RAMPs) 1, 2, and 3 mRNA in these tissues was studied by reverse transcription PCR (RT-PCR). ICC showed that AM2/IMD and AM immunoreactivities were localized in adrenocortical tumors and pheochromocytomas. AM2/IMD and AM immunoreactivities were detected in medulla of attached non-neoplastic tissues, while the degree of immunoreactivity for AM2/IMD and AM in cortices of attached adrenals was relatively weak or undetectable. RIA detected IR-AM2/IMD in adrenal tumors (0.414+/-0.12 to 0.786+/-0.27 pmol/g wet weight, mean+/-S.E.M.) and attached adrenal tissues (0.397+/-0.052 pmol/g wet weight). Reverse-phase high-performance liquid chromatography showed one broad peak eluted in the similar position to synthetic AM2/IMD with several minor peaks. RT-PCR showed expression of AM2/IMD, CRLR, and RAMP1, RAMP2, and RAMP3 mRNA in tissues of adrenal tumors and attached adrenal glands. In conclusion, AM2/IMD is expressed in human adrenal tumors and attached non-neoplastic adrenal tissues and may play (patho-)physiological roles in normal and neoplastic adrenals as an autocrine/paracrine regulator.

  17. Outcome of congenital adrenal hyperplasia.

    PubMed

    Kuhnle, U; Bullinger, M

    1997-09-01

    In congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, affected girls are born with ambiguous genitalia due to increased secretion of androgens in utero by the defective adrenal gland. Even though it is generally accepted that there are differences between male and female brain development, determining factors have been difficult to identify. Girls with CAH have frequently been studied to evaluate the impact of prenatal androgen exposure on psychological, psychosocial, and psychosexual development, and impairments in various areas have been identified. However, there is no comprehensive study available regarding the outcome of this chronic disorder in adult life. We studied the quality of life in women with CAH, with particular emphasis on how they cope with genital malformations, genital operations, and chronic disease as well as lifelong medication. The patients filled out questionnaires covering their physical s