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Sample records for 111in-labelled cholecystokinin-8 cck8

  1. Effect of intraperitoneal and intravenous administration of cholecystokinin-8 and apolipoprotein AIV on intestinal lymphatic CCK-8 and apo AIV concentration.

    PubMed

    Lo, Chun-Min; Xu, Min; Yang, Qing; Zheng, Shuqin; Carey, Katherine M; Tubb, Matthew R; Davidson, W Sean; Liu, Min; Woods, Stephen C; Tso, Patrick

    2009-01-01

    CCK and apolipoprotein AIV (apo AIV) are gastrointestinal satiety signals whose synthesis and secretion by the gut are stimulated by fat absorption. Intraperitoneally administered CCK-8 is more potent in suppressing food intake than a similar dose administered intravenously, but the reason for this disparity is unclear. In contrast, both intravenous and intraperitoneally administered apo AIV are equally as potent in inhibiting food intake. When we compared the lymphatic concentration of CCK-8 and apo AIV, we found that neither intraperitoneally nor intravenously administered CCK-8 or apo AIV altered lymphatic flow rate. Interestingly, intraperitoneal administration of CCK-8 produced a significantly higher lymphatic concentration at 15 min than did intravenous administration. Intraperitoneal injection of apo AIV also yielded a higher lymphatic concentration at 30 min than did intravenous administration. Intraperitoneal administration of CCK-8 and apo AIV also resulted in a much longer period of elevated CCK-8 and apo AIV peptide concentration in lymph than intravenous administration. Furthermore, enzymatic activity of dipeptidyl peptidase IV (DPPIV) and aminopeptidase was higher in plasma than in lymph during fasting, and so, satiation peptides, such as CCK-8 and apo AIV in the lymph, are protected from degradation by the significantly lower DPPIV and aminopeptidase activity levels in lymph than in plasma. Therefore, the higher potency of intraperitoneally administered CCK-8 compared with intravenously administered CCK-8 in inhibiting food intake may be explained by both its higher concentration in lymph and the prolonged duration of its presence in the lamina propria.

  2. Cholecystokinin-8 inhibits methamphetamine-induced neurotoxicity via an anti-oxidative stress pathway.

    PubMed

    Wen, Di; An, Meiling; Gou, Hongyan; Liu, Xia; Liu, Li; Ma, Chunling; Cong, Bin

    2016-12-01

    As a powerful addictive psychostimulant drug, coupled with its neurotoxicity, methamphetamine (METH) abuse may lead to long-lasting abnormalities in brain structure and function. We found that pretreatment of cholecystokinin-8 (CCK-8) inhibited METH-induced brain cellular dopaminergic (DA) damage in the striatum and substantia nigra, and related behavioural deficits and hyperthermia. However, the mechanism of CCK-8 action on METH-induced toxicity is not clear. The aim of this study was to explore whether the possible protective effect of CCK-8 on METH-induced neurotoxicity involved anti-oxidative stress mechanisms. The subtypes of CCK receptors mediating the regulatory action of CCK-8 were also investigated. The present results revealed that CCK-8 dose-dependently inhibited METH-induced cytotoxic effect by activating the CCK2 receptor subtype in PC12 cells and CCK2 receptor stable transfected-HEK293 cells. Pre-treatment of CCK-8 before METH stimulation significantly attenuated the generation of reactive oxygen species and NADPH oxidase activation in PC12 cells. In conclusion, our study demonstrated a protective effect of CCK-8 on METH-induced neurotoxicity in vitro and suggested that a possible mechanism of this action was dependent on the activation of the CCK2 receptor to reduce the neurotoxicity and oxidative stress induced by METH stimulation.

  3. Exogenous cholecystokinin-8 reduces vagal efferent nerve activity in rats through CCKA receptors

    PubMed Central

    Bucinskaite, Violeta; Kurosawa, Mieko; Lundeberg, Thomas

    2000-01-01

    It has been proposed that the vagus nerve plays a role in mediating cholecystokinin-8 (CCK-8) effect on such gastric functions as motility, emptying and gastric acid secretion. To examine the contribution of the efferent pathways in realizing these effects, efferent mass activity in the ventral gastric vagal nerve in Sprague-Dawley rats was recorded.Intravenous infusion of CCK-8 (0.1–1 nmol) suppressed the efferent activity. The effect of CCK-8 was significantly reduced in animals with total subdiaphragmatic vagotomy in comparison to those with partial vagotomy.Intravenous infusion of CCKA receptor antagonist L-364,718 (1–100×10−6 g) blocked the response of vagal efferent activity to 0.1 nmol CCK-8, but the CCKB receptor antagonist L-365,260 (1–100×10−6 g) did not in the conditions of either partial or total vagotomy.Intracisternal infusion of L-364,718 (1×10−6 g) blocked the response of vagal efferent activity to 0.1 nmol CCK-8 i.v.Infusion of exogenous CCK-8 did not affect the activity of supradiaphragmatic vagal afferents.The results suggest that the effect of systemically administered CCK-8 on vagal efferent activity is mediated by both peripherally (subdiaphragmatically) and centrally localized CCKA receptors. PMID:10780970

  4. Protective effects of cholecystokinin-8 on methamphetamine-induced behavioral changes and dopaminergic neurodegeneration in mice.

    PubMed

    Gou, Hongyan; Wen, Di; Ma, Chunling; Li, Ming; Li, Yingmin; Zhang, Wenfang; Liu, Li; Cong, Bin

    2015-04-15

    We investigated whether pretreatment with the neuropeptide cholecystokinin-8 affected methamphetamine (METH)-induced behavioral changes and dopaminergic neurodegeneration in male C57/BL6 mice. CCK-8 pretreatment alone had no effect on locomotion and stereotypic behavior and could not induce behavioral sensitization; however, it attenuated, in a dose-dependent manner, hyperlocomotion and behavioral sensitization induced by a low dose of METH (1mg/kg). CCK-8 attenuated METH-induced stereotypic behavior at a dose of 3mg/kg but not at 10mg/kg. CCK-8 pretreatment attenuated METH (10mg/kg)-induced hyperthermia, the decrease of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum, and TH in the substantia nigra. CCK-8 alone had no effect on rectal temperature, TH and DAT expression in the nigrostriatal region. In conclusion, our study demonstrated that pretreatment with CCK-8 inhibited changes typically induced by repeated exposure to METH, such as hyperlocomotion, behavioral sensitization, stereotypic behavior, and dopaminergic neurotoxicity. These findings make CCK-8 a potential therapeutic agent for the treatment of multiple symptoms associated with METH abuse.

  5. Effect of Heweianshen Decoction on Orexin-A and Cholecystokinin-8 Expression in Rat Models of Insomnia

    PubMed Central

    Liu, Yi; Cao, Ke; Yang, Minghui

    2016-01-01

    Objective. To study the effect of Heweianshen decoction (HAD) on orexin-A and cholecystokinin-8 (CCK-8) expression in rat models of insomnia caused by injecting parachlorophenylalanine (PCPA) intraperitoneally. Methods. Fifty male Wistar rats were randomly divided into five groups (10 rats in each group): blank group, model group, and low-, medium-, and high-dose HAD-treated groups. A rat model of insomnia was established by injecting intraperitoneally with PCPA (300 mg/kg body weight). Rats were given normal saline (10 mL/kg) or 5.25, 10.5, and 21 g/kg HAD by intragastric administration once a day for 6 days. After that, the rats were sacrificed to collect the hypothalamus for tests, using radioimmunoassay to detect the expression of orexin-A and CCK-8. Results. Heweianshen decoction reduced the expression of orexin-A and increased the expression of CCK-8 in the hypothalamus of rat model of insomnia. Conclusion. The therapeutic effect of HAD on insomnia is partially attributed to the decreased expression of orexin-A and increased expression of CCK-8. PMID:27688792

  6. The effects of an essential fatty acid compound and a cholecystokinin-8 antagonist on iron deficiency induced anorexia and learning deficits.

    PubMed

    Yehuda, Shlomo; Mostofsky, David I

    2004-04-01

    Iron deficiency (ID) is among the most common nutritional diseases, causing deleterious effects that include decreases in cognitive function and weight loss. The ID also induces a reduction in the number and affinity of dopaminergic D2 receptors. The new finding that ID induces an increase in the pancreas cells, leads to the hypothesis that cholecystokinin-8 (CCK-8) is involved in the ID effects. The level of CCK-8 was higher among ID rats, compared with normal rats. The ID rats in our study were anorectic and performed poorly in learning tests (Morris water maze and passive avoidance learning). Essential fatty acids (EFA) mediate dopamine activity and have been found to rehabilitate learning deficits. Treatment with a fatty acid compound blocked both the learning deficits and the anorexia, while a CCK-8 antagonist was successful only against the anorectic effects.

  7. CCK-8 prevents the development of kindling and regulates the GABA and NPY expression in the hippocampus of pentylenetetrazole (PTZ)-treated adult rats.

    PubMed

    Tirassa, Paola; Costa, Nicola; Aloe, Luigi

    2005-04-01

    Neuronal loss and irreversible brain damage often cause the worsening of symptoms and the decreased efficacy of pharmacological treatment occurring in epileptic patients and animal models of kindling. Recently we reported that the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) is able to induce the structural and functional neuronal recovery of chemical- and surgical-induced lesions when i.p. injected in rodents. The present study therefore, was aimed at verifying the hypothesis that treatment with a CCK-8 dose having a neuroprotective action might affect brain alterations and the development of kindling in adult rats receiving the convulsant agent pentylenetetrazole (PTZ). Compared to rats receiving Saline prior to PTZ, which manifested clonic-tonic seizures (Class 5 behavioural change scale) after three weeks of treatment, rats pre-treated with CCK-8 showed an improvement of behavioural score exhibiting myoclonus and occasionally tonic seizures (Class 3/4). This decreased susceptibility to develop convulsions was associated with the recovery of PTZ-induced reduction of ChAT levels in forebrain and GABA/GAD expression in the hippocampus. Furthermore, NPY immunoreactivity distribution and NPY mRNA levels were also increased in the hippocampus of rats receiving CCK-8 injection before each PTZ treatment. These data indicate that CCK-8 possesses the ability to prevent and/or suppress the convulsant effects of PTZ by stimulating the synthesis of neurotransmitters/peptides involved in the inhibition of hippocampal hyper-excitability. Our findings suggest that CCK-8 may have anticonvulsant and neuroprotective properties that merit further investigation.

  8. A1-adenosine acute withdrawal response and cholecystokinin-8 induced contractures are regulated by Ca(2+)- and ATP-activated K(+) channels.

    PubMed

    Cascio, Maria Grazia; Valeri, Daniela; Tucker, Steven J; Marini, Pietro

    2015-01-01

    In isolated guinea-pig ileum (GPI), the A1-adenosine acute withdrawal response is under the control of several neuronal signalling systems, including the μ/κ-opioid and the cannabinoid CB1 systems. It is now well established that after the stimulation of the A1-adenosine system, the indirect activation of both μ/κ-opioid and CB1 systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated the involvement of the Ca(2+)/ATP-activated K(+) channels in the regulation of both acute A1-withdrawal and CCk-8-induced contractures in the GPI preparation. Interestingly, we found that: (a) the A1-withdrawal contracture is inhibited by voltage dependent Ca(2+)-activated K(+) channels, Kv, while it is enhanced by the voltage independent Ca(2+)-activated K(+) channels, SKCa; (b) in the presence of CCk-8, the inhibitory effect of the A1 agonist, CPA, on the peptide induced contracture is significantly enhanced by the voltage independent Ca(2+)-activated K(+) channel, SKCa; and (c) the A1-withdrawal contracture precipitated in the presence of CCk-8 is controlled by the ATP-sensitive potassium channels, KATP. Our data suggest, for the first time, that both Ca(2+)- and ATP-activated K(+) channels are involved in the regulation of both A1-withdrawal precipitated and CCk-8 induced contractures.

  9. CCK-8 induces NGF and BDNF synthesis and modulates TrkA and TrkB expression in the rat hippocampus and septum: Effects on kindling development.

    PubMed

    Tirassa, Paola; Costa, Nicola

    2007-01-01

    In our previous studies, we demonstrated that intraperitoneal (i.p.) injections with the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) stimulate the synthesis of the neurotrophin nerve growth factor (NGF) resulting in the structural and functional recovery of neuronal damage. This neurotrophin-mediated neuroprotective action of CCK-8 has opened a new perspective for a better understanding of the CCK neurobiological and pharmacological properties. To explore the possible beneficial effects of the CCK-induced increase of neurotrophin availability in brain, we compared the effects of i.p. CCK-8 in healthy rats and in a chemical kindling model using a subconvulsive dose of pentylenetetrazol (PTZ). Behavioural changes were monitored during treatment and classified according to a six-point scale. After 3 weeks of treatment (12 trials), the PTZ group of rats manifested generalized clonic-tonic seizures (Class 5 behaviour). For this reason, this time point was chosen to compare the effects of CCK-8 treatment on the expression of NGF, the brain derived neurotrophin factor (BDNF) and their receptors in the septum and hippocampus. We found that repeated i.p. injections with CCK-8 in adult rats result in: (1) an increase of NGF and BDNF protein and mRNA levels in the septum and hippocampus; (2) a down-regulation of TrkA and p75NTR and an up-regulation of TrkB; (3) reduced susceptibility to develop chemical kindling; (4) recovery of the PTZ-induced changes in the expression of neurotrophin receptors in the septal and hippocampal tissues. This data clearly indicates that CCK-induced variation of neurotrophin synthesis in brain is able to influence the susceptibility to develop seizures in adult rats most probably by counteracting the progressive neuronal dysfunction and/or damage.

  10. Mercuric chloride-induced gastrin/cholecystokinin 8 immunoreactivity in the central nervous system of the terrestrial slug Semperula maculata: an immunohistochemical study.

    PubMed

    Londhe, Sunil; Kamble, Nitin

    2013-12-01

    We measured the immunoreactivity of the neuropeptide gastrin cholecystokinin 8 (gastrin/CCK 8) in neurons of the terrestrial slug Semperula maculata following acute treatment with mercuric chloride (HgCl2). The distribution of gastrin/CCK 8 was analyzed in neurons of different regions, specifically from cerebral ganglia (procerebrum (pro-c), mesocerebrum (meso-c) and metacerebrum (meta-c). In the control group, neurons of pedal, pleural, parietal and visceral ganglia showed positive immunoreactivity using vertebrate antiserum against gastrin/CCK 8. Gastrin/CCK 8 immunoreactivity was also seen in the fibers and neuropil region of all ganglia. In the cerebral ganglion, 10, 12 and 8 % of the neurons from pro-c, meso-c and meta-c, respectively, were stained with the antibody. The immunostaining was increased in neurons (giant, large, medium and small) after HgCl2 treatment. The treatment greatly increased the mucin content within the neurons. Exposure to HgCl2 enhanced gastrin immunoreactivity in the neurons and this increased with time. Results are discussed in the context of neuropathology in cerebral ganglia associated with the feeding behavior of Semperula maculata.

  11. Antidiabetogenic action of cholecystokinin-8 in type 2 diabetes.

    PubMed

    Ahrén, B; Holst, J J; Efendic, S

    2000-03-01

    Cholecystokinin (CCK) is a gut hormone and a neuropeptide that has the capacity to stimulate insulin secretion. As insulin secretion is impaired in type 2 diabetes, we explored whether exogenous administration of this peptide exerts antidiabetogenic action. The C-terminal octapeptide of CCK (CCK-8) was therefore infused i.v. (24 pmol/kg x h) for 90 min in six healthy postmenopausal women and in six postmenopausal women with type 2 diabetes. At 15 min after start of infusion, a meal was served and ingested during 10 min. On a separate day, saline was infused instead of CCK-8. In both healthy subjects and subjects with type 2 diabetes, CCK-8 reduced the increase in circulating glucose after meal ingestion and potentiated the increase in circulating insulin. The ratio between the area under the curves for serum insulin and plasma glucose during the 15- to 75-min period after meal ingestion was increased by CCK-8 by 198 +/- 18% in healthy subjects (P = 0.002) and by 474 +/- 151% (P = 0.038) in subjects with type 2 diabetes. In contrast, the increase in the circulating levels of gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 (GLP-1), or glucagon after meal ingestion was not significantly affected by CCK-8. The study therefore shows that CCK-8 exerts an antidiabetogenic action in both healthy subjects and type 2 diabetes through an insulinotropic action that most likely is exerted trough a direct islet effect. As at the same time, CCK-8 was infused without any adverse effects, the study suggests that CCK is a potential treatment for type 2 diabetes.

  12. CCK-58 elicits both satiety and satiation in rats while CCK-8 elicits only satiation.

    PubMed

    Overduin, Joost; Gibbs, James; Cummings, David E; Reeve, Joseph R

    2014-04-01

    Reduction of food intake by exogenous cholecystokinin (CCK) has been demonstrated primarily for its short molecular form, CCK-8. Mounting evidence, however, implicates CCK-58 as a major physiologically active CCK form, with different neural and exocrine response profiles than CCK-8. In three studies, we compared meal-pattern effects of intraperitoneal injections CCK-8 vs. CCK-58 in undeprived male Sprague-Dawley rats consuming sweetened condensed milk. In study 1, rats (N=10) received CCK-8, CCK-58 (0.45, 0.9, 1.8 and 3.6 nmol/kg) or vehicle before a 4-h test-food presentation. At most doses, both CCK-8 and CCK-58 similarly reduced meal size relative to vehicle. Meal-size reduction prompted a compensatory shortening of the intermeal interval (IMI) after CCK-8, but not after CCK-58, which uniquely increased the satiety ratio (IMI/size of the preceding meal). In the second study, lick patterns were monitored after administration of 0.9 nmol/kg CCK-58, CCK-8 or vehicle. Lick cluster size, lick efficiency and interlick-interval distribution remained unaltered compared to vehicle, implying natural satiation, rather than illness, following both CCK forms. In study 3, threshold satiating doses of the two CCK forms were given at 5 and 30 min after meal termination, respectively. CCK 58, but not CCK-8 increased the intermeal interval and satiety ratio compared to vehicle. In conclusion, while CCK 58 and CCK-8 both stimulate satiation, thereby reducing meal size, CCK-58 consistently exerts a satiety effect, prolonging IMI. Given the physiological prominence of CCK-58, these results suggest that CCK's role in food intake regulation may require re-examination.

  13. CCK-58 Elicits Both Satiety and Satiation in Rats while CCK-8 Elicits Only Satiation

    PubMed Central

    Overduin, Joost; Gibbs, James; Cummings, David E.; Reeve, Joseph R.

    2014-01-01

    Reduction of food intake by exogenous cholecystokinin (CCK) has been demonstrated primarily for its short molecular form, CCK-8. Mounting evidence, however, implicates CCK-58 as a major physiologically active CCK form, with different neural and exocrine response profiles than CCK-8. In three studies, we compared meal-pattern effects of intraperitoneal injections CCK-8 vs. CCK-58 in undeprived male Sprague-Dawley rats consuming sweetened condensed milk. In study one, rats (N=10) received CCK-8, CCK-58 (0.45, 0.9, 1.8 and 3.6 nmole/kg) or vehicle before a 4-hour test-food presentation. At most doses, both CCK-8 and CCK-58 reduced meal size relative to vehicle. Meal-size reduction prompted a compensatory shortening of the intermeal interval (IMI) after CCK-8, but not after CCK-58, which uniquely increased the satiety ratio (IMI/size of the preceding meal). In the second study, lick patterns were monitored after administration of 0.9nmole/kg CCK-58, CCK-8 or vehicle. Lick cluster size, lick efficiency and interlick-interval distribution remained unaltered compared to vehicle, implying natural satiation, rather than illness, following both CCK forms. In study 3, threshold satiating doses of the two CCK forms were given at 5 and 30 minutes after meal termination, respectively. CCK 58, but not CCK-8 increased the intermeal interval and satiety ratio compared to vehicle. In conclusion, while CCK 58 and CCK-8 both stimulate satiation, thereby reducing meal size, CCK-58 consistently exerts a satiety effect, prolonging IMI. Given the physiological prominence of CCK-58, these results suggest that CCK’s role in food intake regulation may require reexamination. PMID:24468546

  14. Biphasic regulation of the acute μ-withdrawal and CCk-8 contracture responses by the ORL-1 system in guinea pig ileum.

    PubMed

    Marini, Pietro; Romanelli, Luca; Valeri, Daniela; Cascio, Maria Grazia; Tucci, Paolo; Valeri, Pacifico; Palmery, Maura

    2012-01-01

    The cloning of the opioid-receptor-like receptor (ORL-1) and the identification of the orphaninFQ/nociceptin (OFQ/N) as its endogenous agonist has revealed a new G-protein-coupled receptor signalling system. The structural and functional homology of ORL-1 to the opioid receptor systems has posed a number of challenges in the understanding the often competing physiological responses elicited by these G-protein-coupled receptors. We had previously shown that in guinea pig ileum (GPI), the acute μ-withdrawal response is under the inhibitory control of several systems. Specifically, we found that the exposure to a μ-opioid receptor agonist activates indirectly the κ-opioid, the A(1)-adenosine and the cannabinoid CB(1) systems, that in turn inhibit the withdrawal response. The indirect activation of these systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated whether the ORL-1 system is also involved in the regulation of the acute μ-withdrawal response. Interestingly, we found that in GPI preparation, the ORL-1 system is not indirectly activated by the μ-opioid receptor stimulation, but instead the system is able by itself to directly regulate the acute μ-withdrawal response. Moreover, we have demonstrated that the ORL-1 system behaves both as anti-opioid or opioid-like system based on the level of activation. The same behaviour has also been observed in presence of CCk-8. Furthermore, in GPI, the existence of an endogenous tone of the ORL-1 system has been demonstrated. We concluded that the ORL-1 system acts as a neuromodulatory system, whose action is strictly related to the modulation of excitatory neurotrasmitters released in GPI enteric nervous system.

  15. Ethanol exerts dual effects on calcium homeostasis in CCK-8-stimulated mouse pancreatic acinar cells

    PubMed Central

    Fernández-Sánchez, Marcela; del Castillo-Vaquero, Angel; Salido, Ginés M; González, Antonio

    2009-01-01

    Background A significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Nevertheless, the patho-physiological effect of ethanol on pancreatitis remains poorly understood. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca2+ signals in mouse pancreatic acinar cells. Changes in [Ca2+]i and ROS production were analyzed employing fluorescence techniques after loading cells with fura-2 or CM-H2DCFDA, respectively. Results Ethanol, in the concentration range from 1 to 50 mM, evoked an oscillatory pattern in [Ca2+]i. In addition, ethanol evoked reactive oxygen species generation (ROS) production. Stimulation of cells with 1 nM or 20 pM CCK-8, respectively led to a transient change and oscillations in [Ca2+]i. In the presence of ethanol a transformation of 20 pM CCK-8-evoked physiological oscillations into a single transient increase in [Ca2+]i in the majority of cells was observed. Whereas, in response to 1 nM CCK-8, the total Ca2+ mobilization was significantly increased by ethanol pre-treatment. Preincubation of cells with 1 mM 4-MP, an inhibitor of alcohol dehydrogenase, or 10 μM of the antioxidant cinnamtannin B-1, reverted the effect of ethanol on total Ca2+ mobilization evoked by 1 nM CCK-8. Cinnamtannin B-1 blocked ethanol-evoked ROS production. Conclusion ethanol may lead, either directly or through ROS generation, to an over stimulation of pancreatic acinar cells in response to CCK-8, resulting in a higher Ca2+ mobilization compared to normal conditions. The actions of ethanol on CCK-8-stimulation of cells create a situation potentially leading to Ca2+ overload, which is a common pathological precursor that mediates pancreatitis. PMID:19878551

  16. Peripherally injected CCK-8S activates CART positive neurons of the paraventricular nucleus in rats

    PubMed Central

    Noetzel, Steffen; Inhoff, Tobias; Goebel, Miriam; Taché, Yvette; Veh, Rüdiger W.; Bannert, Norbert; Grötzinger, Carsten; Wiedenmann, Bertram; Klapp, Burghard F.; Mönnikes, Hubert; Kobelt, Peter

    2014-01-01

    Cholecystokinin (CCK) plays a role in the short-term inhibition of food intake. Cocaine- and amphetamine-regulated transcript (CART) peptide has been observed in neurons of the paraventricular nucleus (PVN). It has been reported that intracerebroventricular injection of CART peptide inhibits food intake in rodents. The aim of the study was to determine whether intraperitoneally (ip) injected CCK-8S affects neuronal activity of PVN-CART neurons. Ad libitum fed male Sprague-Dawley rats received 6 or 10 μg/kg CCK-8S or 0.15 M NaCl ip (n = 4/group). The number of c-Fos-immunoreactive neurons was determined in the PVN, arcuate nucleus (ARC), and the nucleus of the solitary tract (NTS). CCK-8S dose-dependently increased the number of c-Fos-immunoreactive neurons in the PVN (mean ± SEM: 102 ± 6 vs. 150 ± 5 neurons/section, p < 0.05) and compared to vehicle treated rats (18 ± 7, p < 0.05 vs. 6 and 10 μg/kg CCK-8S). CCK-8S at both doses induced an increase in the number of c-Fos-immunoreactive neurons in the NTS (65 ± 13, p < 0.05, and 182 ± 16, p < 0.05). No effect on the number of c-Fos neurons was observed in the ARC. Immunostaining for CART and c-Fos revealed a dose-dependent increase of activated CART neurons (19 ± 3 vs. 29 ± 7; p < 0.05), only few activated CART neuron were observed in the vehicle group (1 ± 0). The present observation shows that CCK-8S injected ip induces an increase in neuronal activity in PVN-CART neurons and suggests that CART neurons in the PVN may play a role in the mediation of peripheral CCK-8S's anorexigenic effects. PMID:20307613

  17. Effects of cholecystokinin-8 on morphine-induced spatial reference memory impairment in mice.

    PubMed

    Yang, Shengchang; Wen, Di; Dong, Mei; Li, Dong; Sun, Donglei; Ma, Chunling; Cong, Bin

    2013-11-01

    Acute and chronic exposure to opiate drugs impaired various types of memory processes. To date, there is no preventive treatment for opiate-induced memory impairment and the related mechanism is still unclear. CCK-8 is the most potent endogenous anti-opioid peptide and has been shown to exert memory-enhancing effect, but the effect of CCK-8 on morphine-induced memory impairment has not been reported. By using Morris water maze, we found that escape latency to the hidden platform in navigation test was not influenced, but performance in the probe test was seriously poor in morphine dependency mice. Amnesia induced by chronic morphine treatment was significantly alleviated by pre-treatment with CCK-8 (0.01, 0.1 and 1 μg, i.c.v.), and CCK-8 (0.1 and 1 μg, i.c.v.) treatment alone could improve performance in either navigation or probe test. Furthermore, Golgi-Cox staining analysis revealed that pre-treatment with CCK-8 (1 μg, i.c.v.) reversed spine density decreased in CA1 region of hippocampus in morphine dependency mice, and CCK-8 (1 μg, i.c.v.) alone obviously increased spine density in CA1. Our findings conclude spine density change in CA1 region of hippocampus may be the structural plasticity mechanism which is responsible for enhancing effect of CCK-8 on spatial reference memory. Therefore, CCK-8 could effectively improve memory impairment in morphine dependency mice.

  18. Cholecystokinin-8 suppressed /sup 3/H-etorphine binding to rat brain opiate receptors

    SciTech Connect

    Wang, X.J.; Fan, S.G.; Ren, M.F.; Han, J.S.

    1989-01-01

    Radioreceptor assay (RRA) was adopted to analyze the influence of CCK-8 on /sup 3/H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 suppressed the binding of /sup 3/H-etorphine. This effect was completely reversed by proglumide at 1/mu/M. Rosenthal analysis for saturation revealed two populations of /sup 3/H-etorphine binding sites. CCK-8 inhibited /sup 3/H-etorphine binding to the high affinity sites by an increase in Kd and decrease in Bmax without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 was also completely reversed by proglumide at 1/mu/M. Unsulfated CCK-8 produced only a slight increase in Kd of the high affinity sites without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

  19. sup 111 In-labeled nonspecific immunoglobulin scanning in the detection of focal infection

    SciTech Connect

    Rubin, R.H.; Fischman, A.J.; Callahan, R.J.; Khaw, B.A.; Keech, F.; Ahmad, M.; Wilkinson, R.; Strauss, H.W. )

    1989-10-05

    We performed radionuclide scanning after the intravenous injection of human IgG labeled with indium-111 in 128 patients with suspected focal sites of inflammation. Localization of 111In-labeled IgG correlated with clinical findings in 51 infected patients (21 with abdominal or pelvic infections, 11 with intravascular infections, 7 with pulmonary infections, and 12 with skeletal infections). Infecting organisms included gram-positive bacteria, gram-negative bacteria, Pneumocystis carinii, Mycoplasma pneumoniae, and Candida albicans. No focal localization of 111In-labeled IgG was observed in 63 patients without infection. There were five false negative results, and nine results were unusable. Serial scans were carried out in eight patients: continued localization correctly predicted relapse in six, and the absence of localization indicated resolution in two. To determine whether 111In-labeled IgG localization was specific for inflammation, we studied 16 patients with cancer. Focal localization occurred in 13 of these patients (5 with melanomas, 5 with gynecologic cancers, and 1 each with lymphoma, prostate cancer, and malignant fibrous histiocytoma). No localization was seen in patients with renal or colon cancer or metastatic medullary carcinoma of the thyroid. We conclude that 111In-labeled IgG imaging is effective for the detection of focal infection and that serial scans may be useful in assessing therapeutic efficacy. This technique may also be helpful in the evaluation of certain cancers.

  20. Facilitation of amphetamine-induced hypothermia in mice by GABA agonists and CCK-8.

    PubMed

    Boschi, G; Launay, N; Rips, R

    1991-04-01

    1. Amphetamine-induced hypothermia in mice is facilitated by dopaminergic stimulation and 5-hydroxytryptaminergic inhibition. The present study was designed to investigate: (a) the involvement of other neuronal systems, such as the gamma-aminobutyric acid (GABA), the opioid and the cholecystokinin (CCK-8) systems; (b) the possible contribution of hydroxylated metabolites of amphetamine to the hypothermia; (c) the capacity of dopamine itself to induce hypothermia and its mechanisms, in order to clarify the resistance of amphetamine-induced hypothermia to certain neuroleptics. 2. Pretreatment with the GABA antagonists, bicuculline and picrotoxin, did not inhibit amphetamine-induced hypothermia. The GABAB agonist, baclofen (2.5 mg kg-1, i.p.) potentiated this hypothermia, whereas the GABAA agonist, muscimol, did not. gamma-Butyrolactone (GBL) (40 mg kg-1, i.p.) and the neuropeptide CCK-8 (0.04 mg kg-1, i.p.) also induced potentiation. The opioid antagonist, naloxone, was without effect. 3. Dopamine itself (3, 9, 16 and 27 micrograms, i.c.v.) induced less hypothermia than the same doses of amphetamine. Sulpiride did not block dopamine-induced hypothermia, but pimozide (4 mg kg-1, i.p.), cis(z)flupentixol (0.25 mg kg-1, i.p.) and haloperidol (5 micrograms, i.c.v.) did. The direct dopamine receptor agonist, apomorphine, did not alter the hypothermia. Neither the 5-hydroxytryptamine (5-HT) receptor blocker, cyproheptadine, nor the inhibitor of 5-HT synthesis, p-chlorophenylalanine (PCPA), modified dopamine-induced hypothermia. Fluoxetine, an inhibitor of 5-HT reuptake, had no effect, whereas quipazine (6 mg kg-1, i.p.), a 5-HT agonist, totally prevented the hypothermia. Hypothermia was unaffected by pretreatment with CCK-8. 4. These data indicate that the hypothermia induced by amphetamine involves not only dopaminergic and 5-hydroxytryptaminergic systems which are functionally antagonistic, but is also facilitated by direct or indirect GABA and CCK-8 receptor stimulation

  1. Characterization of the receptors and mechanisms involved in the cardiovascular actions of sCCK-8 in the pithed rat.

    PubMed Central

    Gaw, A. J.; Hills, D. M.; Spraggs, C. F.

    1995-01-01

    1. The cardiovascular actions of cholecystokinin and related peptides were investigated in the pithed rat. The receptors and the mechanisms involved in these experiments were characterized. 2. Sulphated cholecystokinin octapeptide (sCCK-8, 0.1-100 nmol kg-1, i.v.) elicited a dose-dependent bradycardia and increase in mean arterial blood pressure. Neither gastrin-17 nor pentagastrin had any effect at concentrations up to 100 nmol kg-1. 3. Both the pressor response and bradycardia elicited by sCCK-8 were reduced by the selective CCKA receptor antagonists, devazepide (0.5-50 nmol kg-1) and lorglumide (1-7 mumol kg-1). The selective CCKB receptor antagonists, CI-988 (1 mumol kg-1) and L-365,260 (15 mumol kg-1) did not inhibit the effects of sCCK-8. 4. The pressor response induced with sCCK-8 was reduced by treatment with either phentolamine (3 mumol kg-1) or guanethidine (2 mumol kg-1) and was unaffected by treatment with propranolol, atropine or hexamethonium. The pressor response also persisted following bilateral adrenalectomy. 5. The bradycardia induced with sCCK-8 was unaffected by treatment with phentolamine, propranolol, guanethidine, atropine, hexamethonium or bilateral adrenalectomy. 6. The tetrapeptide of cholecystokinin (CCK-4) elicited a dose-dependent pressor response but did not induce bradycardia. The pressor response was unaffected by devazepide (50 nmol kg-1), L-365260 (15 mumol kg-1) or phentolamine (3 mumol kg-1). 7. In the pithed rat, sCCK-8 acted via CCKA receptors to increase arterial blood pressure indirectly, at least in part, through activation of alpha-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582487

  2. CCK-58 prolongs the intermeal interval, whereas CCK-8 reduces this interval: not all forms of cholecystokinin have equal bioactivity.

    PubMed

    Sayegh, Ayman I; Washington, Martha C; Raboin, Shannon J; Aglan, Amnah H; Reeve, Joseph R

    2014-05-01

    It has been accepted for decades that "all forms of cholecystokinin (CCK) have equal bioactivity," despite accumulating evidence to the contrary. To challenge this concept, we compared two feeding responses, meal size (MS, 10% sucrose) and intermeal interval (IMI), in response to CCK-58, which is the major endocrine form of CCK, and CCK-8, which is the most abundantly utilized form. Doses (0, 0.1, 0.5, 0.75, 1, 3 and 5 nmol/kg) were administered intraperitoneally over a 210-min test to Sprague Dawley rats that had been food-deprived overnight. We found that (1) all doses of CCK-58, except the lowest dose, and all doses of CCK-8, except the lowest two doses, reduced food intake more than vehicle did; (2) at two doses, 0.75 and 3 nmol/kg, CCK-58 increased the IMI, while CCK-8 failed to alter this feeding response; and (3) CCK-58, at all but the lowest two doses, increased the satiety ratio (IMI between first and second meals (min) divided by first MS (ml)) relative to vehicle, while CCK-8 did not affect this value. These findings demonstrate that the only circulating form of CCK in rats, CCK-58, prolongs the IMI more than CCK-8, the peptide generally utilized in feeding studies. Taken together, these results add to a growing list of functions where CCK-8 and CCK-58 express qualitatively different bioactivities. In conclusion, the hypothesis that "all forms of cholecystokinin (CCK) have equal bioactivity" is not supported.

  3. Sulfated cholecystokinin-8 activates phospho-mTOR immunoreactive neurons of the paraventricular nucleus in rats

    PubMed Central

    Frommelt, Lisa; Inhoff, Tobias; Lommel, Reinhardt; Stengel, Andreas; Taché, Yvette; Grötzinger, Carsten; Bannert, Norbert; Wiedenmann, Bertram; Klapp, Burghard F.; Kobelt, Peter

    2014-01-01

    The serin/threonin-kinase, mammalian target of rapamycin (mTOR) was detected in the arcuate nucleus (ARC) and paraventricular nucleus of the hypothalamus (PVN) and suggested to play a role in the integration of satiety signals. Since cholecystokinin (CCK) plays a role in the short-term inhibition of food intake and induces c-Fos in PVN neurons, the aim was to determine whether intraperitoneally injected CCK-8S affects the neuronal activity in cells immunoreactive for phospho-mTOR in the PVN. Ad libitum fed male Sprague-Dawley rats received 6 or 10 μg/kg CCK-8S or 0.15 M NaCl ip (n=4/group). The number of c-Fosimmunoreactive (ir) neurons was assessed in the PVN, ARC and in the nucleus of the solitary tract (NTS). CCK-8S increased the number of c-Fos-ir neurons in the PVN (6 μg: 103 ± 13 vs. 10 μg: 165 ± 14 neurons/section; p<0.05) compared to vehicle treated rats (4 ± 1, p<0.05), but not in the ARC. CCK-8S also dose-dependently increased the number of c-Fos neurons in the NTS. Staining for phospho-mTOR and c-Fos in the PVN showed a dose-dependent increase of activated phospho-mTOR neurons (17 ± 3 vs. 38 ± 2 neurons/section; p<0.05), while no activated phospho-mTOR neurons were observed in the vehicle group. Triple staining in the PVN showed activation of phospho-mTOR neurons co-localized with oxytocin, corresponding to 9.8 ± 3.6% and 19.5 ± 3.3% of oxytocin neurons respectively. Our observations indicate that peripheral CCK-8S activates phospho-mTOR neurons in the PVN and suggest that phospho-mTOR plays a role in the mediation of CCK-8S's anorexigenic effects. PMID:20933028

  4. Ileal interposition attenuates the satiety responses evoked by cholecystokinin-8 and -33.

    PubMed

    Metcalf, Shannon A; Washington, Martha C; Brown, Thelma A L; Williams, Carol S; Strader, April D; Sayegh, Ayman I

    2011-06-01

    One of the possible mechanisms by which the weight-reducing surgical procedure ileal interposition (II) works is by increasing circulating levels of lower gut peptides that reduce food intake, such as glucagon like peptide-1 and peptide YY. However, since this surgery involves both lower and upper gut segments, we tested the hypothesis that II alters the satiety responses evoked by the classic upper gut peptide cholecystokinin (CCK). To test this hypothesis, we determined meal size (MS), intermeal interval (IMI) and satiety ratio (SR) evoked by CCK-8 and -33 (0, 1, 3, 5nmol/kg, i.p.) in two groups of rats, II and sham-operated. CCK-8 and -33 reduced MS more in the sham group than in the II group; CCK-33 prolonged IMI in the sham group and increased SR in both groups. Reduction of cumulative food intake by CCK-8 in II rats was blocked by devazepide, a CCK(1) receptor antagonist. In addition, as previously reported, we found that II resulted in a slight reduction in body weight compared to sham-operated rats. Based on these observations, we conclude that ileal interposition attenuates the satiety responses of CCK. Therefore, it is unlikely that this peptide plays a significant role in reduction of body weight by this surgery.

  5. Five Stabilized 111In-labeled neurotensin analogs in nude mice bearing HT29 tumors.

    PubMed

    Janssen, Paul J J M; de Visser, Moniaue; Verwijnen, Suzanne M; Bernard, Bert F; Srinivasan, Ananth; Erion, Jack L; Breeman, Wouter A P; Vulto, Arnold G; Krenning, Eric P; de Jong, Marion

    2007-06-01

    Neurotensin (NT) receptors are overexpressed in different human tumors, such as human ductal pancreatic adenocarcinoma. New stable neurotensin analogs with high receptor affinity have been synthesized by replacing arginine residues with lysine and arginine derivatives. The aim of this study was to explore the biodistribution, tumor uptake, kidney localization, and stability characteristics of these new analogs in order to develop new diagnostic tools for exocrine pancreatic cancer. Four (111)In-labeled DTPA-chelated NT analogs and one (111)In-labeled DOTA-chelated NT analog were evaluated in NMRI nude mice bearing NT receptor-positive HT29 tumors. Experiments with a coinjection of unlabeled NT or lysine were performed to investigate receptor-mediated uptake and kidney protection, respectively. In addition, the in vivo serum stability of the most promising analog was analyzed. In the biodistribution study in mice, at 4 hours postinjection, a low percentage of the injected dose per gram (%ID/g) of tissue for all compounds was found in NT receptor-negative organs, such as the blood, spleen, pancreas, liver, muscle, and femur. A high uptake was found in the colon, intestine, kidneys, and in implanted HT29 tumors. The coinjection of excess unlabeled neurotensin significantly reduced tumor uptake, showing tumor uptake to be receptor-mediated. To a lesser extent, this was also observed for the colon, but not for other tissues. We concluded that DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH and the DOTA-linked counterpart have the most favorable biodistribution properties regarding tumor uptake.

  6. 111In-labelled octreotide binding by the somatostatin receptor subtype 2 in neuroendocrine tumours.

    PubMed

    Hashemi, S H; Benjegård, S-A; Ahlman, H; Wängberg, B; Forssell-Aronsson, E; Billig, H; Nilsson, O

    2003-05-01

    The aim of this study was to investigate the importance of somatostatin receptor subtype 2 (SSTR2) expression for 111In-labelled diethylenetriamine-pentaacetic acid (DTPA)-D-Phe1-octreotide binding and uptake of 111In in neuroendocrine tumours. 111In activity concentrations in surgical biopsies from neuroendocrine tumours (midgut carcinoid and medullary thyroid carcinoma), breast carcinoma and blood were determined 1-8 days after intravenous injection of 111In-labelled DTPA-D-Phe1-octreotide (140-350 MBq). The ratio of 111In activity concentrations between tumour tissue and blood (T/B value) was calculated. The expression of SSTR2 messenger RNA (mRNA) in tumour biopsies was quantitated by ribonuclease protection assay and SSTR2 protein was localized by immunocytochemistry. T/B values were highest for tumour biopsies from midgut carcinoids (mean 160 (range 4-1200); n = 65) followed by medullary thyroid carcinoma (mean 38 (range 2-350); n = 88) and breast carcinoma (mean 18 (range 4-41); n = 4). The expression of SSTR2 mRNA (relative to the NCI-H69 cell line) was highest in tumour biopsies from midgut carcinoids (mean 2.5 (range 0.83-6.0); n = 40) followed by medullary thyroid carcinoma (mean 1.3 (range 0.20-6.0); n = 7) and breast carcinoma (mean 0.66 (range 0.29-1.0); n = 9). In tumour biopsies SSTR2 protein was localized exclusively to tumour cells. Midgut carcinoid tumours showed a much higher level of SSTR2 expression than medullary thyroid carcinoma in accordance with superior tumour imaging by octreotide scintigraphy. The high SSTR2 mRNA values and T/B values observed in midgut carcinoid tumours were positively correlated. Copyright 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

  7. Presynaptically mediated effects of cholecystokinin-8 on the excitability of area postrema neurons in rat brain slices.

    PubMed

    Sugeta, Shingo; Hirai, Yoshiyuki; Maezawa, Hitoshi; Inoue, Nobuo; Yamazaki, Yutaka; Funahashi, Makoto

    2015-08-27

    Cholecystokinin (CCK) is a well-known gut hormone that shows anorexigenic effects via action at peripheral and central receptors. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. The area postrema is one of the circumventricular organs, located on the dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. Blood vessels in the area postrema lack a blood brain barrier, offering specific central neural elements unique access to circulating substances. Immunohistochemical studies show CCK-A receptors in the area postrema, and we reported CCK-sensitive area postrema neurons. However, the receptive mechanism of CCK in area postrema neurons still remains unexplained. We investigated the responses of area postrema neurons to agonists and antagonists of CCK receptors using whole cell and perforated patch-clamp recordings in rat brain slices. The application of CCK-8 elicited excitatory responses, such as increases in the frequency of mEPSCs (miniature excitatory postsynaptic currents), a shift toward larger amplitude mEPSCs, and increases in the frequency of action potentials. These changes were found mostly in cells not displaying the hyperpolarization-activated cation current (Ih), except for small excitatory changes in a minority of Ih-positive neurons. Tonic inward currents or an inhibitory response to CCK-8 were never seen. Analysis of the amplitude of mEPSCs before and after the administration of CCK-8 indicated the responses mediated via the presynaptic receptors. The effect of CCK-8 was abolished in the presence of CNQX (AMPA type glutamate receptor antagonist). In the presence of lorglumide (a selective CCK-A receptor antagonist), CCK-8-induced excitatory responses were inhibited. No cells responded to the administration of non-sulfated CCK-8 (CCK-8NS, a selective CCK-B receptor agonist). We conclude that CCK-8 exerts its action via presynaptic CCK-A receptors

  8. Studies on the influence of CCK-8 on the ability of obestatin to reduce food intake, gain in body weight and related lipid parameters.

    PubMed

    Nagaraj, ShreeRanga; Manjappara, Uma V

    2016-06-01

    In an effort to mimic in part the redundancy of satiety peptides involved in energy homeostasis, the combined benefits of the well-established satiety peptide CCK8 and an apparently anorectic peptide obestatin were studied in Swiss albino mice. The optimal dose of obestatin that was required to give the most pronounced effect with CCK8 was worked out by varying the concentration of obestatin while keeping CCK8 concentration constant at 200 nmol/KgBW. Mice administered 160 nmol obestatin and 200 nmol CCK8 per kilogram body weight showed the most drastic reduction in food intake. Gain in body weight was arrested after day four during the eight day experiment. These studies reemphasize the beneficial effects imparted by co-administration of obestatin and CCK8 and their potential use towards countering obesity.

  9. Relative distribution of gastrin-, CCK-8-, NPY- and CGRP-immunoreactive cells in the digestive tract of dorado (Salminus brasiliensis).

    PubMed

    Pereira, R T; Costa, L S; Oliveira, I R C; Araújo, J C; Aerts, M; Vigliano, F A; Rosa, P V

    2015-04-01

    The endocrine cells (ECs) of the gastrointestinal mucosa form the largest endocrine system in the body, not only in terms of cell numbers but also in terms of the different produced substances. Data describing the association between the relative distributions of the peptide-specific ECs in relation to feeding habits can be useful tools that enable the creation of a general expected pattern of EC distribution. We aimed to investigate the distribution of ECs immunoreactive for the peptides gastrin (GAS), cholecystokinin (CCK-8), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) in different segments of the digestive tract of carnivorous fish dorado (Salminus brasiliensis) by using immunohistochemistry procedures. The distribution of endocrine cells immunoreactive for gastrin (GAS), cholecystokinin (CCK-8), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) in digestive tract of dorado S. brasiliensis was examined by immunohistochemistry. The results describe the association between the distribution of the peptide-specific endocrine cells and feeding habits in different carnivorous fish. The largest number of endocrine cells immunoreactive for GAS, CCK-8, and CGRP were found in the pyloric stomach region and the pyloric caeca. However, NPY-immunoreactive endocrine cells were markedly restricted to the midgut. The distribution pattern of endocrine cells identified in S. brasiliensis is similar to that found in other carnivorous fishes.

  10. Preparation, cytotoxicity, and in vivo antitumor efficacy of 111In-labeled modular nanotransporters

    PubMed Central

    Slastnikova, Tatiana A; Rosenkranz, Andrey A; Morozova, Natalia B; Vorontsova, Maria S; Petriev, Vasiliy M; Lupanova, Tatiana N; Ulasov, Alexey V; Zalutsky, Michael R; Yakubovskaya, Raisa I; Sobolev, Alexander S

    2017-01-01

    Purpose Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting 111In for cancer therapy. Methods Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with 111In in high specific activity. Cytotoxicity of the 111In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted 111In-labeled MNT in B16-F1 melanoma tumor-bearing mice. Results The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of 111In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111In by serial imaging and significant tumor growth delay (82% growth inhibition) were found. Conclusion The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted 111In-NOTA–MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted 111In-NOTA–MNT suggests further applications of the MNT delivery strategy should be explored. PMID:28138237

  11. Sulfated Cholecystokinin-8 Promotes CD36—Mediated Fatty Acid Uptake into Primary Mouse Duodenal Enterocytes

    PubMed Central

    Demenis, Claire; McLaughlin, John; Smith, Craig P.

    2017-01-01

    Cholecystokinin (CCK) is an archetypal incretin hormone secreted by intestinal enteroendocrine cells (EEC) in response to ingested nutrients. The aim of this study was to determine whether CCK modulates enterocyte fatty acid uptake by primary mouse duodenal cells. Exposure of primary mouse duodenal cells to 10 pM sulfated CCK-8 caused a two fold increase in dodecanoic acid fatty acid (FA) uptake. The selective CCK A receptor antagonist loxiglumide (100 μM) completely abolished the CCK-8 induced FA uptake. The CD36 fatty acid translocase-specific inhibitor sulfo-N-succinimidyl oleate (1 μM) also completely inhibited CCK-8 induced FA uptake, as did treatment with 200 μM phloretin. Together these data show CCK induces FA uptake into duodenal enterocytes; this action involves the CCK-RA receptor and is carrier mediated by CD36.

  12. Cholecystokinin-8 activates myenteric neurons in 21- and 35-day old but not 4- and 14-day old rats.

    PubMed

    Washington, Martha C; Murry, Candace R; Raboin, Shannon J; Roberson, Allison E; Mansour, Mahmoud M; Williams, Carol S; Sayegh, Ayman I

    2011-02-01

    Cholecystokinin (CCK) activates the myenteric neurons of adult rats. The goal of this work is to determine the ontogeny of this activation by CCK-8 in the myenteric plexus of the duodenum (2cm immediately following the pyloric sphincter aborally) and compare it with that of the dorsal vagal complex (DVC) - which occurs in 1-day old pups. Despite the existence of both of the CCK receptors, CCK(1) and CCK(2), in 4, 14, 21 and 35 day old rats, CCK-8 (0, 5, 10, 20 and 40μg/kg, i.p.) increased Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of 21- and 35-day old rats but in the DVC of all age groups. As such, this belated activation of myenteric neurons by CCK-8 compared to the DVC may reflect a delayed role for these neurons in CCK-related functions.

  13. Influences of CCK-8 on expressions of apoptosis-related genes in prefrontal cortex neurons of morphine-relapse rats.

    PubMed

    Ye, Guanghua; Tao, Luyang; Ma, Chunling; Wen, Di; Yu, Linsheng; Fan, Yanyan; Hu, Haiyan; Chen, Xiping; Chu, Yang; Gao, Yuan; Gao, Cheng; Wang, Haochen

    2016-09-19

    In order to elucidate the influences of CCK-8 on expressions of apoptosis-related genes, Bax, Bcl-2 and Caspase-3, of prefrontal cortex neurons in morphine-relapse rats, an effective, successful morphine-relapse-rat model using the conditioned place preference (CPP) under CCK-8 (0.01, 0.1 and 1.0μg, i.c.v) intervention was established. The prefrontal cortexes were made into slices with the cellular plasmas immunohistochemically stained. The expressions of Bax, Bcl-2, Caspase-3 of neurons were evaluated through their scores, and each corresponding ratio of Bax and Bcl-2 (Bax/Bcl-2) was also computed. The results showed that the expression of Bcl-2 was very weak and those of Bax and Caspase-3 were hardly seen in group normal saline; the expressions of Bax and Caspase-3 were strong and that of Bcl-2 was weak in group morphine and compared to group normal saline, there were significant differences (P<0.05); the expressions of Bax, Caspase-3 and the ratios of Bax/Bcl-2 have a gradually-decreased trend in the sequence of group 0.01μg, group 0.1μg and group 1.0μg, but the expression of Bcl-2 has an opposite trend in the same sequence, and compared to group morphine, there were significant differences (P<0.05) excluding group 0.01μg. So we draw a conclusion that CCK-8 (0.1 and 1.0μg, i.c.v) could protect neurons of prefrontal cortex through up-regulating the expression of Bcl-2, down-regulating those of Bax and Caspase-3 and reducing Bax/Bcl-2 ratio in the model of morphine-relapse rats. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Detection of acute inflammation with /sup 111/In-labeled nonspecific polyclonal IgG

    SciTech Connect

    Fischman, A.J.; Rubin, R.H.; Khaw, B.A.; Callahan, R.J.; Wilkinson, R.; Keech, F.; Nedelman, M.; Dragotakes, S.; Kramer, P.B.; LaMuraglia, G.M.

    1988-10-01

    The detection of focal sites of inflammation is an integral part of the clinical evaluation of the febrile patient. When anatomically distinct abscesses are present, lesion detection can be accomplished by standard radiographic techniques, particularly in patients with normal anatomy. At the phlegmon stage, however, and in patients who have undergone surgery, these techniques are considerably less effective. While radionuclide methods, such as Gallium-67 (67Ga)-citrate and Indium-111 (111In)-labeled WBCs have been relatively successful for the detection of early inflammation, neither approach is ideal. In the course of studies addressing the use of specific organism-directed antibodies for imaging experimental infections in animals, we observed that nonspecific polyclonal immunoglobulin G (IgG) localized as well as specific antibodies. Preliminary experiments suggested that the Fc portion of IgG is necessary for effective inflammation localization. Since polyclonal IgG in gram quantities has been safely used for therapy in patients with immune deficiency states, we decided to test whether milligram quantities of radiolabeled IgG could image focal sites of inflammation in humans. Thus far, we have studied a series of 84 patients with suspected lesions in the abdomen, pelvis, vascular grafts, lungs, or bones/joints. In 48 of 52 patients with focal lesions detected by surgery, computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound (US), the IgG scan correctly localized the site, while 31 patients without focal inflammation had no abnormal focal localization of the radiopharmaceutical. Four patients had false negative scans and one patient had a false positive scan. For this small series, the overall sensitivity and specificity were 92% and 95%, respectively. In this report, we review our experience with this exciting new agent.

  15. Cholecystokinin B-type receptors mediate a G-protein-dependent depolarizing action of sulphated cholecystokinin ocatapeptide (CCK-8s) on rodent neonatal spinal ventral horn neurons.

    PubMed

    Oz, Murat; Yang, Keun-Hang; Shippenberg, Toni S; Renaud, Leo P; O'Donovan, Michael J

    2007-09-01

    Reports of cholecystokinin (CCK) binding and expression of CCK receptors in neonatal rodent spinal cord suggest that CCK may influence neuronal excitability. In patch-clamp recordings from 19/21 ventral horn motoneurons in neonatal (PN 5-12 days) rat spinal cord slices, we noted a slowly rising and prolonged membrane depolarization induced by bath-applied sulfated CCK octapeptide (CCK-8s; 1 microM), blockable by the CCK B receptor antagonist L-365,260 (1 microM). Responses to nonsulfated CCK-8 or CCK-4 were significantly weaker. Under voltage clamp (V H -65 mV), 22/24 motoneurons displayed a CCK-8s-induced tetrodotoxin-resistant inward current [peak: -136 +/- 28 pA] with a similar time course, mediated via reduction in a potassium conductance. In 29/31 unidentified neurons, CCK-8s induced a significantly smaller inward current (peak: -42.8 +/- 5.6 pA), and I-V plots revealed either membrane conductance decrease with net inward current reversal at 101.3 +/- 4.4 mV (n = 16), membrane conductance increase with net current reversing at 36.1 +/- 3.8 mV (n = 4), or parallel shift (n = 9). Intracellular GTP-gamma-S significantly prolonged the effect of CCK-8s (n = 6), whereas GDP-beta-S significantly reduced the CCK-8s response (n = 6). Peak inward currents were significantly reduced after 5-min perfusion with N-ethylmaleimide. In isolated neonatal mouse spinal cord preparations, CCK-8s (30-300 nM) increased the amplitude and discharge of spontaneous depolarizations recorded from lumbosacral ventral roots. These observations imply functional postsynaptic G-protein-coupled CCK B receptors are prevalent in neonatal rodent spinal cord.

  16. Peripheral injected cholecystokinin-8S modulates the concentration of serotonin in nerve fibers of the rat brainstem.

    PubMed

    Engster, Kim-Marie; Frommelt, Lisa; Hofmann, Tobias; Nolte, Sandra; Fischer, Felix; Rose, Matthias; Stengel, Andreas; Kobelt, Peter

    2014-09-01

    Serotonin and cholecystokinin (CCK) play a role in the short-term inhibition of food intake. It is known that peripheral injection of CCK increases c-Fos-immunoreactivity (Fos-IR) in the nucleus of the solitary tract (NTS) in rats, and injection of the serotonin antagonist ondansetron decreases the number of c-Fos-IR cells in the NTS. This supports the idea of serotonin contributing to the effects of CCK. The aim of the present study was to elucidate whether peripherally injected CCK-8S modulates the concentration of serotonin in brain feeding-regulatory nuclei. Ad libitum fed male Sprague-Dawley rats received 5.2 and 8.7 nmol/kg CCK-8S (n=3/group) or 0.15M NaCl (n=3-5/group) injected intraperitoneally (ip). The number of c-Fos-IR neurons, and the fluorescence intensity of serotonin in nerve fibers were assessed in the paraventricular nucleus (PVN), arcuate nucleus (ARC), NTS and dorsal motor nucleus of the vagus (DMV). CCK-8S increased the number of c-Fos-ir neurons in the NTS (mean±SEM: 72±4, and 112±5 neurons/section, respectively) compared to vehicle-treated rats (7±2 neurons/section, P<0.05), but did not modulate c-Fos expression in the DMV or ARC. Additionally, CCK-8S dose-dependently increased the number of c-Fos-positive neurons in the PVN (218±15 and 128±14, respectively vs. 19±5, P<0.05). In the NTS and DMV we observed a decrease of serotonin-immunoreactivity 90 min after injection of CCK-8S (46±2 and 49±8 pixel/section, respectively) compared to vehicle (81±8 pixel/section, P<0.05). No changes of serotonin-immunoreactivity were observed in the PVN and ARC. Our results suggest that serotonin is involved in the mediation of CCK-8's effects in the brainstem.

  17. Multimodality Molecular Imaging of [18F]-Fluorinated Carboplatin Derivative Encapsulated in [111In]-Labeled Liposomes

    NASA Astrophysics Data System (ADS)

    Lamichhane, Narottam

    -(5-fluoro-pentyl)-2-methyl malonic acid as the labeling agent to coordinate with the cisplatin aqua complex. It was then used to treat various cell lines and compared with cisplatin and carboplatin at different concentrations ranging from 0.001 microM to 100 microM for 72 hrs and 96 hrs. IC50 values calculated from cell viability indicated that 19F-FCP is a more potent drug than Carboplatin. Manual radiosynthesis and characterization of [18F]-FCP was performed using [18F]-2-(5-fluoro-pentyl)-2-methyl malonic acid with coordination with cisplatin aqua complex. Automated radiosynthesis of [18F]-FCP was optimized using the manual synthetic procedures and using them as macros for the radiosynthesizer. [18F]-FCP was evaluated in vivo with detailed biodistribution studies and PET imaging in normal and KB 3-1 and KB 8-5 tumor xenograft bearing nude mice. The biodistribution studies and PET imaging of [18F]-FCP showed major uptake in kidneys which attributes to the renal clearance of radiotracer. In vivo plasma and urine stability demonstrated intact [18F]-FCP. [ 111In]-Labeled Liposomes was synthesized and physiochemical properties were assessed with DLS. [111In]-Labeled Liposome was evaluated in vivo with detailed pharmacokinetic studies and SPECT imaging. The biodistribution and ROI analysis from SPECT imaging showed the spleen and liver uptake of [111In]-Labeled Liposome and subsequent clearance of activity with time. [18F]-FCP encapsulated [111In]-Labeled Liposome was developed and physiochemical properties were characterized with DLS. [18F]-FCP encapsulated [111In]-Labeled Liposome was used for in vivo dual tracer PET and SPECT imaging from the same nanoconstruct in KB 3-1 (sensitive) and COLO 205 (resistant) tumor xenograft bearing nude mice. PET imaging of [18F]-FCP in KB 3-1 (sensitive) and COLO 205 (resistant) tumor xenograft bearing nude mice was performed. Naked [18F]-FCP and [18F]-FCP encapsulated [ 111In]-Labeled Liposome showed different pharmacokinetic profiles. PET

  18. Stabilised 111In-labelled DTPA- and DOTA-conjugated neurotensin analogues for imaging and therapy of exocrine pancreatic cancer.

    PubMed

    de Visser, M; Janssen, P J J M; Srinivasan, A; Reubi, J C; Waser, B; Erion, J L; Schmidt, M A; Krenning, E P; de Jong, M

    2003-08-01

    Neurotensin (NT) receptors are overexpressed in exocrine pancreatic cancer and Ewing's sarcoma. The potential utility of native NT in cancer diagnosis and therapy is, however, limited by its rapid degradation in vivo. Therefore, NT analogues were synthesised with modified lysine and arginine derivatives to enhance stability and coupled either to DTPA, to enable high specific activity labelling with indium-111 for imaging, or to DOTA, to enable high specific activity labelling with beta-emitting radionuclides, such as lutetium-177 and yttrium-90. Based on serum stability (4 h incubation at 37 degrees C in human serum) and receptor binding affinity, the five most promising analogues were selected and further evaluated in in vitro internalisation studies in human colorectal adenocarcinoma HT29 cells, which overexpress NT receptors. All five NT analogues bound with high affinity to NT receptors on human exocrine pancreatic tumour sections. The analogues could be labelled with (111)In to a high specific activity. The (111)In-labelled compounds were found to be very stable in serum. Incubation of HT29 cells with the (111)In-labelled analogues at 37 degrees C showed rapid receptor-mediated uptake and internalisation. The most promising analogue, peptide 2530 [DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH] was further tested in vivo in a biodistribution study using HT29 tumour-bearing nude mice. The results of this study showed low percentages of injected dose per gram tissue of this (111)In-labelled 2530 analogue in receptor-negative organs like blood, spleen, pancreas, liver, muscle and femur. Good uptake was found in the receptor-positive HT29 tumour and high uptake was present in the kidneys. Co-injection of excess unlabelled NT significantly reduced tumour uptake, showing that tumour uptake is a receptor-mediated process. With their enhanced stability, maintained high receptor affinity and rapid receptor-mediated internalisation, the (111)In-labelled DTPA

  19. Administration of cholecystokinin sulphated octapeptide (CCK-8S) induces changes on rat amino acid tissue levels and on a behavioral test for anxiety.

    PubMed

    Acosta, G B

    1998-10-01

    1. The effect of the intraperitoneal administration of cholecystokinin sulphated octapeptide (CCK-8S) (10 nmol/kg i.p.) on endogenous levels of several amino acids in five areas of the rat brain was analyzed. The olfactory bulb, hypothalamus, hippocampus, cerebral frontal cortex, and corpus striatum were evaluated. In addition, the effects of CCK-8S and PD 135,158 (1 mg/kg), a selective CCK(B) antagonist, on the performance of rats submitted to a dark/light transition test were also studied. 2. Upon administration of CCK-8S, the concentration of glutamate was reduced (27%) in the olfactory bulb. The same was observed when the levels of glycine (31%) or alanine (43%) were determined. No significant effects were produced by CCK-8S on cortical and hypothalamic levels. In the hippocampus, the concentration of both glutamate (27%) and taurine (29%) were reduced, whereas the levels of GABA in the striatum (29%) were increased. 3. After a single injection of CCK-8S, the time spent by the rats in the illuminated site of the dark/light transition test box, was not changed. On the contrary, the administration of PD 135,158 increased the time spent in the lighted compartment. 4. These results show that systemic administration of CCK-8S produced regional specific changes in brain amino acids, without producing any significant behavioral modification in the rat exposed to a dark/light box. In contrast, the selective CCKB receptor antagonist, PD 135,158, induces anxiolytic-like action in an animal model of anxiety.

  20. Linker effects on biological properties of 111In-labeled DTPA conjugates of a cyclic RGDfK dimer.

    PubMed

    Jia, Bing; Liu, Zhaofei; Shi, Jiyun; Yu, Zilin; Yang, Zhi; Zhao, Huiyun; He, Zhengjie; Liu, Shuang; Wang, Fan

    2008-01-01

    In this report, we present in vitro and in vivo evaluation of three 111 In-labeled DTPA conjugates of a cyclic RGDfK dimer: DTPA-Bn-SU016 (SU016 = E[c(RGDfK)] 2; DTPA-Bn = 2-( p-isothioureidobenzyl)diethylenetriaminepentaacetic acid), DTPA-Bn-E-SU016 ( E = glutamic acid) and DTPA-Bn-Cys-SU016 (Cys = cysteic acid). The integrin alpha vbeta 3 binding affinities of SU016, DTPA-Bn-SU016, DTPA-Bn-E-SU016, and DTPA-Bn-Cys-SU016 were determined to be 5.0 +/- 0.7 nM, 7.9 +/- 0.6 nM, 5.8 +/- 0.6 nM, and 6.9 +/- 0.9 nM, respectively, against 125 I-c(RGDyK) in binding to integrin alpha vbeta3, suggesting that E or Cys residue has little effect on the integrin alpha vbeta3 affinity of E[c(RGDfK)] 2. It was also found that the 111 In-labeling efficiency of DTPA-Bn-SU016 and DTPA-Bn-E-SU016 is 3-5 times better than that of DOTA analogues due to fast chelation kinetics and high-yield 111 In-labeling under mild conditions (e.g., room temperature). Biodistribution studies were performed using BALB/c nude mice bearing U87MG human glioma xenografts. 111 In-DTPA-Bn-SU016, 111 In-DTPA-Bn-E-SU016, and 111 In-DTPA-Bn-Cys-SU016 all displayed rapid blood clearance. Their tumor uptake was comparable between 0.5 and 4 h postinjection (p.i.) within experimental error. 111 In-DTPA-Bn-E-SU016 had a significantly lower ( p < 0.01) kidney uptake than 111 In-DTPA-Bn-SU016 and 111 In-DTPA-Bn-Cys-SU016. The liver uptake of 111 In-DTPA-Bn-SU016 was 1.69 +/- 0.18% ID/g at 24 h p.i., while the liver uptake values of 111 In-DTPA-Bn-E-SU016 and 111 In-DTPA-Bn-Cys-SU016 were 0.55 +/- 0.11% ID/g and 0.79 +/- 0.15% ID/g at 24 h p.i., respectively. Among the three 111 In radiotracers evaluated in this study, 111 In-DTPA-Bn-E-SU016 has the lowest liver and kidney uptake and the best tumor/liver and tumor/kidney ratios. Results from metabolism studies indicated that there is little metabolism (<10%) for three 111 In radiotracers at 1 h p.i. Imaging data showed that tumors can be clearly visualized at 4 h p

  1. Linker Effects on Biological Properties of 111In-Labeled DTPA Conjugates of a Cyclic RGDfK Dimer

    PubMed Central

    Jia, Bing; Liu, Zhaofei; Shi, Jiyun; Yu, Zilin; Yang, Zhi; Zhao, Huiyun; He, Zhengjie; Liu, Shuang; Wang, Fan

    2008-01-01

    In this report, we present in vitro and in vivo evaluation of three 111In-labeled DTPA conjugates of a cyclic RGDfK dimer: DTPA-Bn-SU016 (SU016 = E[c(RGDfK)]2; DTPA-Bn = 2-(p-isothiocyanobenzyl)diethylenetriaminepentaacetic acid), DTPA-Bn-E-SU016 (E = glutamic acid) and DTPA-Bn-Cys-SU016 (Cys = cysteic acid). The integrin αvβ3 binding affinities of SU016, DTPA-Bn-SU016, DTPA-Bn-E-SU016 and DTPA-Bn-Cys-SU016 were determined to be 5.0 ± 0.7 nM, 7.9 ± 0.6 nM, 5.8 ± 0.6 nM and 6.9 ± 0.9 nM, respectively, against 125I-c(RGDyK) in binding to integrin αvβ3, suggesting that E or Cys residue has little effect on the integrin αvβ3 affinity of E[c(RGDfK)]2. It was also found that the 111In-labeling efficiency of DTPA-Bn-SU016 and DTPA-Bn-E-SU016 is 3–5 times better than that of DOTA analogs due to fast chelation kinetics and high-yield 111In-labeling under mild conditions (e.g. room temperature). Biodistribution studies were performed using BALB/c nude mice bearing U87MG human glioma xenografts. 111In-DTPA-Bn-SU016, 111In-DTPA-Bn-E-SU016 and 111In-DTPA-Bn-Cys-SU016 all displayed a rapid blood clearance. Their tumor uptake was comparable between 0.5 h and 4 h postinjection (p.i.) within experimental error. 111In-DTPA-Bn-E-SU016 had a significantly lower (p < 0.01) kidney uptake than 111In-DTPA-Bn-SU016 and 111In-DTPA-Bn-Cys-SU016. The liver uptake of 111In-DTPA-Bn-SU016 was 1.69 ± 0.18 %ID/g at 24 h p.i. while the liver uptake of 111In-DTPA-Bn-E-SU016 and 111In-DTPA-Bn-Cys-SU016 was 0.55 ± 0.11 %ID/g and 0.79 ± 0.15 %ID/g at 24 h p.i., respectively. Among the three 111In radiotracers evaluated in this study, 111In-DTPA-Bn-E-SU016 has the lowest liver and kidney uptake and the best tumor/liver and tumor/kidney ratios. Results from metabolism studies indicated that there is little (<10%) metabolism for three 111In radiotracers at 1 h p.i. Imaging data showed that tumors can be clearly visualized at 4 h p.i. with good contrast in the tumor-bearing mice administered

  2. Effects of peripherally administered cholecystokinin-8 and secretin on feeding/drinking and oxytocin-mRFP1 fluorescence in transgenic rats.

    PubMed

    Motojima, Yasuhito; Kawasaki, Makoto; Matsuura, Takanori; Saito, Reiko; Yoshimura, Mitsuhiro; Hashimoto, Hirofumi; Ueno, Hiromichi; Maruyama, Takashi; Suzuki, Hitoshi; Ohnishi, Hideo; Sakai, Akinori; Ueta, Yoichi

    2016-08-01

    Peripheral administration of cholecystokinin (CCK)-8 or secretin activates oxytocin (OXT)-secreting neurons in the hypothalamus. Although OXT is involved in the regulation of feeding behavior, detailed mechanism remains unclear. In the present study, we examined the central OXTergic pathways after intraperitoneally (i.p.) administration of CCK-8 and secretin using male OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats and male Wistar rats. I.p. administration of CCK-8 (50μg/kg) and secretin (100μg/kg) decreased food intake in these rats. While i.p. administration of CCK-8 decreased water intake, i.p. administration of secretin increased water intake. Immunohistochemical study revealed that Fos-Like-Immunoreactive cells were observed abundantly in the brainstem and in the OXT neurons in the dorsal division of the parvocellular paraventricular nucleus (dpPVN). We could observe marked increase of mRFP1 fluorescence, as an indicator for OXT, in the dpPVN and mRFP1-positive granules in axon terminals of the dpPVN OXT neurons in the nucleus tractus solitarius (NTS) after i.p. administration of CCK-8 and secretin. These results provide us the evidence that, at least in part, i.p. administration of CCK-8 or secretin might be involved in the regulation of feeding/drinking via a OXTergic pathway from the dpPVN to the NTS.

  3. Anxiogenic-like action of caerulein, a CCK-8 receptor agonist, in the mouse: influence of acute and subchronic diazepam treatment.

    PubMed

    Harro, J; Põld, M; Vasar, E

    1990-01-01

    Effects of caerulein, a cholecystokinin octapeptide (CCK-8) receptor agonist, on exploratory activity of mice were investigated. Exploratory and locomotor activity of animals were measured using elevated plus-maze and open field tests. The systemic administration of caerulein at non-sedative doses (100 ng/kg-1 micrograms/kg i.p.) resulted in a significant decrease in the exploratory activity of mice. This effect was completely blocked by proglumide, a CCK-8 receptor. Acute treatment with low doses (0.1-0.75 mg/kg i.p.) of diazepam did not attenuate the anxiogenic-like effect of caerulein, but at more high doses of diazepam the coadministration depressed locomotor activity in mice. After subchronic diazepam treatment (2.5 mg/kg once a day, 10 days, i.p.) tolerance was developed toward the sedative effect of diazepam, and 72 h after withdrawal of the drug the animals showed increased anxiety in the plus-maze test. 30 min after the last injection procedure the anxiogenic-like effect of caerulein (500 ng/kg i.p.) on exploration was absent in both diazepam or vehicle groups. However, 72 h after the last pretreatment injection caerulein (500 ng/kg i.p.) reduced significantly the exploratory activity in control group, whereas it was inactive after diazepam withdrawal. The results obtained in this study support the hypothesis that endogenous CCK-8 an CCK-8 receptors are involved in the neurochemistry of anxiety and the anxiolytic action of benzodiazepine tranquillizers.

  4. The effect of ibuprofen on accumulation of /sup 111/In-labeled platelets and leukocytes in experimental myocardial infarction

    SciTech Connect

    Romson, J.L.; Hook, B.G.; Rigot, V.H.; Schork, M.A.; Swanson, D.P.; Lucchesi, B.R.

    1982-11-01

    To assess the ability of ibuprofen to influence the extent of platelet aggregation and leukocyte infiltration during acute myocardial infarction, autologous indium-111 (/sup 111/In)-labeled platelets or leukocytes were injected before 60 minutes of left circumflex coronary artery (LCx) occlusion, followed by 24 hours of reperfusion in the canine heart. Myocardial infarct size, as a percent of the area at risk, was reduced in the ibuprofen-treated group (12.5 mg/kg i.v. every 4 hours beginning 30 minutes before LCx occlusion) by 40%, from 48 +/- 4% in control animals to 29 +/- 4% in ibuprofen-treated dogs (p . 0.005). Quantification of the platelet-associated /sup 111/In radioactivity in irreversibly injured myocardium indicated that ibuprofen did not alter the accumulation of platelets in infarcted myocardium. In contrast, leukocyte accumulation in infarcted tissue was reduced significantly. In tissue samples with 0.41-0.60 gram infarct, the infarcted/normal ratio of leukocyte radioactivity was 12 +/- 2 in control dogs and 4 +/- 1 in ibuprofen-treated dogs, which represents a 67% reduction in leukocyte accumulation in ibuprofen-treated compared with control dogs. Similar reductions were found in other gram-infarct-weight categories. Although both platelets and leukocytes accumulate in infarcted canine myocardium, ibuprofen may exert its beneficial effect on ischemic myocardium by suppressing the inflammatory response associated with myocardial ischemia and infarction.

  5. (pGlu-Gln)-CCK-8[mPEG]: a novel, long-acting, mini-PEGylated cholecystokinin (CCK) agonist that improves metabolic status in dietary-induced diabetes.

    PubMed

    Irwin, Nigel; Frizelle, Pamela; O'Harte, Finbarr P M; Flatt, Peter R

    2013-08-01

    Cholecystokinin (CCK) is a gastrointestinal hormone that has been proposed as a potential therapeutic option for obesity-diabetes. As such, (pGlu-Gln)-CCK-8 is an N-terminally modified CCK-8 analogue with improved biological effectiveness over the native peptide. The current study has examined the in vitro stability, biological activity and in vivo therapeutic applicability of a novel second generation mini-PEGylated form of (pGlu-Gln)-CCK-8, (pGlu-Gln)-CCK-8[mPEG]. (pGlu-Gln)-CCK-8[mPEG] was completely resistant to enzymatic degradation and in addition displayed similar insulinotropic (p<0.05 to p<0.001) and satiating effects (p<0.01 to p<0.001) as (pGlu-Gln)-CCK-8. This confirmed the capability of (pGlu-Gln)-CCK-8[mPEG] to bind to and activate the CCK receptor. Sub-chronic twice daily injection of (pGlu-Gln)-CCK-8[mPEG] in high fat fed mice for 35days significantly decreased body weight gain (p<0.05), food intake (p<0.01 to p<0.001) and triacylglycerol deposition in liver (p<0.001) and muscle (p<0.001). Furthermore, (pGlu-Gln)-CCK-8[mPEG] markedly improved intraperitoneal glucose tolerance (p<0.05) and insulin sensitivity (p<0.001). Despite this therapeutic profile, once daily injection of (pGlu-Gln)-CCK-8[mPEG] in high fat fed mice for 33days, at the same dose, was not associated with alterations in food intake and body weight. In addition, metabolic responses to exogenous glucose and insulin injection were similar to saline treated controls. These studies emphasise the therapeutic potential of (pGlu-Gln)-CCK-8[mPEG] and similar molecules. A more detailed analysis of the dose and administration schedule employed for (pGlu-Gln)-CCK-8[mPEG] could provide a novel and effective compound to treat obesity-diabetes. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Synthesis and opioid receptor binding of indium (III) and [(111)In]-labeled macrocyclic conjugates of diprenorphine: novel ligands designed for imaging studies of peripheral opioid receptors.

    PubMed

    Srivastava, Shefali; Fergason-Cantrell, Emily A; Nahas, Roger I; Lever, John R

    2016-10-06

    Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to μ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [(111)In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (~99%) and high specific radioactivity (>4000 mCi/μmol). Their log D7.4 values were -2.21 to -1.66. In comparison, DPN is lipophilic, with a log D7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [(111)In]-labeled DPN-DOTA conjugates for imaging trials.

  7. Synthesis and opioid receptor binding of indium (III) and [111In]-labeled macrocyclic conjugates of diprenorphine: novel ligands designed for imaging studies of peripheral opioid receptors

    PubMed Central

    Srivastava, Shefali; Fergason-Cantrell, Emily A.; Nahas, Roger I.; Lever, John R.

    2016-01-01

    Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to μ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [111In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (~99%) and high specific radioactivity (>4000 mCi/μmol). Their log D7.4 values were −2.21 to −1.66. In comparison, DPN is lipophilic, with a log D7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [111In]-labeled DPN-DOTA conjugates for imaging trials. PMID:28190898

  8. Effect of Chelator Conjugation Level and Injection Dose on Tumor and Organ Uptake of 111In Labeled MORAb-009, an Anti-mesothelin Antibody

    PubMed Central

    Shin, I. S.; Lee, S.-M.; Kim, H. S.; Yao, Z.; Regino, C.; Sato, N.; Cheng, K. T.; Hassan, R.; Campo, M. F.; Albone, E. F.; Choyke, P. L.; Pastan, I.; Paik, C. H.

    2012-01-01

    Introduction Radiolabeling of a monoclonal antibody (mAb) with a metallic radionuclide requires the conjugation of a bifunctional chelator to the mAb. The conjugation, however, can alter the physical and immunological properties of the mAb, consequently affecting its tumor targeting pharmacokinetics. In this study, we investigated the effect of the amount of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) conjugated to MORAb-009, a mAb directed against mesothelin and the effect of MORAb dose on the biodistribution of 111In labeled MORAb-009. Methods We used nude mice bearing A431/K5 tumor as a mesothelin-positive tumor model and A431 tumor as a mesothelin-negative control. To find the optimal level of CHX-A″ conjugation, CHX-A″-MORAb-009 conjugates with 2.4, 3.5, and 5.5 CHX-A″ molecules were investigated. To investigate the effect of injected MORAb-009 dose on neutralizing the shed-mesothelin in the circulation, the biodistribution studies were performed after the i.v. co-injection of the 111In labeled MORAb-009 (2.4 CHX-A″/MORAb-009) with three different doses, 0.2, 2, and 30 μg of MORAb-009. Results The tumor uptake in A431/K5 tumor was 4 times higher than that in A431 tumor, indicating that the tumor uptake in A431/K5 was mesothelin-mediated. The conjugate with 5.5 CHX-A″ showed a lower isoelectric point (pI) and lower immunoreactivity (IR) than the 2.4 CHX-A″ conjugate. These differences were reflected in biodistribution of the 111In label. The 111In labeled MORAb-009 conjugated with 2.4 CHX-A″ produced higher tumor uptake, and lower liver and spleen uptakes than the 5.5 CHX-A″ conjugate. The biodistribution studies also revealed that the tumor uptake was significantly affected by the injected MORAb-009 dose and tumor size. The 30 μg dose produced higher tumor uptake than the 0.2 and 2 μg doses whereas the 30 μg dose produced lower liver and spleen uptakes than the 0.2 μg dose. Conclusion This study

  9. Satiety enhancement by selective orexin-1 receptor antagonist SB-334867: influence of test context and profile comparison with CCK-8S.

    PubMed

    Ishii, Y; Blundell, J E; Halford, J C G; Upton, N; Porter, R; Johns, A; Rodgers, R J

    2005-05-07

    Acute systemic treatment with the selective orexin-1 (OX1R) antagonist SB-334867 reduces food intake in rats, an effect associated with an acceleration in behavioural satiety and unrelated to gross behavioural disruption, alterations in palatability, or toxicity. However, as enhanced satiety is behaviourally indexed by an earlier-than-normal transition from eating to resting, and since orexin-A has been implicated in mechanisms of arousal, it remains possible that sedation contributes to the anorectic effect of acute OX1R blockade. Previous work has shown that, when treated with SB-334867 (30 mg/kg, i.p.) 30 min before a 1h test with palatable food, rats begin to show appreciable levels of resting 10-15 min earlier than under control conditions (i.e. around 20 min versus 30-35 min into the session). The present results demonstrate that a 20 min increase in the injection-test interval (i.e. 50 min) had no significant impact on the anorectic, behavioural or weight gain effects of SB-334867 in non-deprived male rats. Most importantly, this altered treatment regimen led to a temporal profile of resting virtually identical to that previously observed with the more conventional 30 min injection-test interval. Although parallel studies indicated that the OX1R antagonist accelerated the onset of resting (and suppressed most active behaviours) even in the absence of food, an equianorectic dose of the natural satiety-related signal cholescystokinin octapeptide (CCK-8S; 5 microg/kg, i.p.) also produced very similar behavioural effects regardless of the presence of food. Together with evidence that SB-334867 preserves the structural integrity of natural feeding behaviour, does not induce nausea/illness or alter taste/palatability and fails to influence EEG measures of arousal/sleep, the present findings are consistent with the view that acute OX1R antagonism selectively enhances satiety. However, unlike the immediate short-circuiting of the satiety sequence induced by CCK-8S

  10. Plasma concentrations of cholecystokinin, CCK-8, and CCK-33, 39 in rats, determined by a method based on enzyme digestion of gastrin before HPLC and RIA detection of CCK.

    PubMed Central

    Lindén, A; Carlquist, M; Hansen, S; Uvnäs-Moberg, K

    1989-01-01

    A new specific method for determination of cholecystokinin, CCK-8, and CCK-33, 39 in rat plasma is described. Plasma CCK radioimmunoassay (RIA) is difficult, because of cross-reactivity with gastrin. In the rat, problems because of difficulties in separating gastrin from CCK by high performance liquid chromatography (HPLC) exist. These were solved by enzyme digestion of gastrin before HPLC separation of molecular variants of CCK from gastrin fragments. Cholecystokinin immunoreactive forms in the HPLC fractions were determined by an antibody, which recognises the carboxyl terminus of CCK and gastrin. Fasting concentrations of small (CCK-8) and large (CCK-33, 39) molecular forms of CCK averaged 1.9 (0.3) pM and were raised to 13.4 (3.8) pM in rats fed ad libitum. Cholecystokinin in lactating rats rose two-fold after suckling, compared with 2.8 fold in response to feeding. The basal ratio between CCK-8 and CCK-33, 39 was approximately 1:1, but increased in favour of CCK-8 after feeding and in response to suckling. Gastrin like immunoreactivity measured in unextracted plasma was found to rise after feeding, but was unchanged in response to suckling. PMID:2703143

  11. Utility of 111In-labelled leucocyte scintigraphy in patients with fever of unknown origin in an era of changing disease spectrum and investigational techniques.

    PubMed

    Seshadri, Nagabhushan; Solanki, Chandra K; Balan, Kottekkattu

    2008-03-01

    (111)In-labelled leucocyte, imaging is often used to investigate patients with fever of unknown origin (FUO). Its diagnostic performance, however, has been variable and a broad range of sensitivities and specificities have been reported. The purpose of this investigation was to evaluate the usefulness of (111)In-labelled leucocytes scintigraphy in the detection of a cause of FUO in the light of a changing spectrum of diseases causing it and advances in investigational techniques. Sixty-one patients with a clinical diagnosis of FUO underwent whole-body (111)In-troponolate-labelled leucocyte scintigraphy in our department over a 2-year period between February 2004 and February 2006. Of these, 54 patients were retrospectively reviewed to identify a cause of FUO. Other parameters such as C-reactive protein (CRP), leucocyte count and radiological findings were also evaluated. Leucocyte scintigraphy was found to be true positive in 12 patients, true negative in 24 patients, false positive in 10 patients and false negative in eight patients. The overall sensitivity of scintigraphy was 60%, specificity 71%, positive predictive value 55%, and negative predictive value 75%. There was no difference in the scintigraphic sensitivity between patients with spontaneous FUO and those with post-operative FUO although the latter showed a higher specificity and PPV. CRP and leucocyte count did not differ significantly between patients with true positive and true negative scintigrams. Overall, 83% of patients with abnormal radiological examinations had positive findings on scintigraphy and 87% of patients with negative findings on radiology had normal scintigraphy. Despite changes in disease spectrum and advances in investigational techniques, our results suggest that (111)In-leucocyte scintigraphy is still a useful technique in establishing the cause of FUO. A higher PPV of this test in post-operative situations makes it especially applicable in this category of patients. Equally, the

  12. A high-fat diet raises fasting plasma CCK but does not affect upper gut motility, PYY, and ghrelin, or energy intake during CCK-8 infusion in lean men.

    PubMed

    Little, Tanya J; Feltrin, Kate L; Horowitz, Michael; Meyer, James H; Wishart, Judith; Chapman, Ian M; Feinle-Bisset, Christine

    2008-01-01

    There is evidence from studies in animals that the effects of both fat and CCK on gastrointestinal function and energy intake are attenuated by consumption of a high-fat diet. In humans, the effects of exogenous CCK-8 on antropyloroduodenal motility, plasma CCK, peptide YY (PYY), and ghrelin concentrations, appetite, and energy intake are attenuated by a high-fat diet. Ten healthy lean males consumed isocaloric diets (~15,400 kJ per day), containing either 44% (high-fat, HF) or 9% (low-fat, LF) fat, for 21 days in single-blind, randomized, cross-over fashion. Immediately following each diet (i.e., on day 22), subjects received a 45-min intravenous infusion of CCK-8 (2 ng.kg(-1).min(-1)), and effects on antropyloroduodenal motility, plasma CCK, PYY, ghrelin concentrations, hunger, and fullness were determined. Thirty minutes after commencement of the infusion, subjects were offered a buffet-style meal, from which energy intake (in kilojoules) was quantified. Body weight was unaffected by the diets. Fasting CCK (P < 0.05), but not PYY and ghrelin, concentrations were greater following the HF, compared with the LF, diet. Infusion of CCK-8 stimulated pyloric pressures (P < 0.01) and suppressed antral and duodenal pressures (P < 0.05), with no difference between the diets. Energy intake also did not differ between the diets. Short-term consumption of a HF diet increases fasting plasma CCK concentrations but does not affect upper gut motility, PYY and ghrelin, or energy intake during CCK-8 infusion, in a dose of 2 ng.kg(-1).min(-1), in healthy males.

  13. Biodistribution, pharmacokinetics, and nuclear imaging studies of 111In-labeled rGel/BLyS fusion toxin in SCID mice bearing B cell lymphoma.

    PubMed

    Wen, Xiaoxia; Lyu, Mi-Ae; Zhang, Rui; Lu, Wei; Huang, Qian; Liang, Dong; Rosenblum, Michael G; Li, Chun

    2011-08-01

    We examined the biodistribution and pharmacokinetics of (111)In-labeled rGel/BLyS, a gelonin toxin (rGel)-B lymphocyte stimulator (BLyS) fusion protein. rGel/BLyS was labeled with In-111 through DTPA with a labeling efficiency >95%. Biodistribution/imaging studies were obtained in severe-combined immunodeficiency mice bearing diffuse large B cell lymphoma OCI-Ly10. Pharmacokinetic studies were performed in BALB/c mice. In vitro, DTPA-conjugated rGel/BLyS displayed selective cytotoxicity against OCI-Ly10 cells and mantle cell lymphoma JeKo cells. In vivo, rGel/BLyS exhibited a tri-exponential disposition with a rapid initial mean distribution followed by an extensive mean distribution and a long terminal elimination phase. At 48 h after injection, uptake of the radiotracer in tumors was 1.25 %ID/g, with a tumor-to-blood ratio of 13. Tumors were clearly visualized at 24-72 h post-injection. Micro-SPECT-CT images and ex vivo analyses confirmed the accumulation of rGel/BLyS in OCI-Ly10 tumors. (111)In-DTPA-rGel/BLyS are distributed to B cell tumors and induce apoptosis in tumors. Preclinical antitumor studies using rGel/BLyS should use a twice-per-week treatment schedule.

  14. Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr-labeled platelets differ

    SciTech Connect

    Heyns A du, P.; Badenhorst, P.N.; Loetter, M.G.P.; Pieters, H.; Wessels, P.; Kotze, H.F.

    1986-01-01

    Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr-labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.

  15. /sup 111/In-labeled platelets: effects of heparin on uptake by venous thrombi and relationship to the activated partial thromboplastin time

    SciTech Connect

    Fedullo, P.F.; Moser, K.M.; Moser, K.S.; Konopka, R.; Hartman, M.T.

    1982-09-01

    The goal of heparin therapy in deep vein thrombosis is to prevent thrombus extension. The relationship between thrombus extension and the results of coagulation tests used to monitor heparin therapy is unclear. To explore this relationship, we studied the effect of several heparin regimens on the accretion of /sup 111/In-labeled platelets on fresh venous thrombi, as detected by gamma imaging, and monitored the activated partial thromboplastin time (APTT). Six dogs were treated with a 300-U/kg bolus of heparin followed by a 90-U/kg/hour heparin infusion, a dose of heparin sufficient to increase the APTT to levels greater than eight times baseline (APTT ratio); platelet accretion (thrombus imaging) occurred only after the heparin effect was reversed with protamine sulfate. Nineteen dogs were treated with a 150-U/kg bolus of heparin followed by a 4-hour, 45-U/kg/hour heparin infusion; a thrombus was demonstrated only after protamine injection in 12 (mean APTT ratio 1.3 +/- 0.19) and before protamine injection in seven. In thirteen of these 19 dogs, 30 minutes separated the platelet injection from heparin therapy, while in six this duration was less than 30 minutes. In four of these six dogs, thrombi were demonstrated before protamine therapy and at APTT ratios greater than 3.0. Finally, 10 dogs were treated with a 100-U/kg bolus followed by a 3-hour, 50-U/kg/hour heparin infusion, after which the APTT was allowed to return to baseline values spontaneously. In all 10 dogs, a thrombus was demonstrated only after cessation of the heparin infusion, and at a mean APTT ratio of 1.4 +/- 0.15 times baseline. These results suggest that, except with very early platelet injection, platelet accretion by thrombi is consistently inhibited by heparin at APTT ratios greater than 2.5. Platelet accretion by venous thrombi occurs within narrow limits of heparin effect as reflected by the APTT.

  16. Differential effects of predosing on tumor and tissue uptake of an 111In-labeled anti-TENB2 antibody-drug conjugate.

    PubMed

    Boswell, C Andrew; Mundo, Eduardo E; Zhang, Crystal; Stainton, Shannon L; Yu, Shang-Fan; Lacap, Jennifer A; Mao, Weiguang; Kozak, Katherine R; Fourie, Aimee; Polakis, Paul; Khawli, Leslie A; Lin, Kedan

    2012-09-01

    TENB2, also known as tomoregulin or transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is a transmembrane proteoglycan overexpressed in human prostate tumors. This protein is a promising target for antimitotic monomethyl auristatin E (MMAE)-based antibody-drug conjugate (ADC) therapy. Nonlinear pharmacokinetics in normal mice suggested that antigen expression in normal tissues may contribute to targeted mediated disposition. We evaluated a predosing strategy with unconjugated antibody to block ADC uptake in target-expressing tissues in a mouse model while striving to preserve tumor uptake and efficacy. Unconjugated, unlabeled antibody was preadministered to mice bearing the TENB2-expressing human prostate explant model, LuCaP 77, followed by a single administration of (111)In-labeled anti-TENB2-MMAE for biodistribution and SPECT/CT studies. A tumor-growth-inhibition study was conducted to determine the pharmacodynamic consequences of predosing. Preadministration of anti-TENB2 at 1 mg/kg significantly increased blood exposure of the radiolabeled ADC and reduced intestinal, hepatic, and splenic uptake while not affecting tumor accretion. Similar tumor-to-heart ratios were measured by SPECT/CT at 24 h with and without the predose. Consistent with this, the preadministration of 0.75 mg/kg did not interfere with efficacy in a tumor-growth study dosed at 0.75 mg or 2.5 mg of ADC per kilogram. Overall, the potential to mask peripheral, nontumor antigen uptake while preserving tumor uptake and efficacy could ameliorate toxicity and may significantly affect future dosing strategies for ADCs.

  17. Role of the amygdaloid cholecystokinin (CCK)/gastrin-2 receptors and terminal networks in the modulation of anxiety in the rat. Effects of CCK-4 and CCK-8S on anxiety-like behaviour and [3H]GABA release.

    PubMed

    Pérez de la Mora, Miguel; Hernández-Gómez, Ana María; Arizmendi-García, Yexel; Jacobsen, Kirsten X; Lara-García, Daniel; Flores-Gracia, Candy; Crespo-Ramírez, Minerva; Gallegos-Cari, Andrea; Nuche-Bricaire, Avril; Fuxe, Kjell

    2007-12-01

    The amygdala plays a key role in fear and anxiety. The intercalated islands are clusters of glutamate-responsive GABAergic neurons rich in cholecystokinin (CCK)-2 receptors which control the trafficking of nerve impulses from the cerebral cortex to the central nucleus of amygdala. In this study, the nature of the CCK-glutamate-GABA interactions within the rat rostral amygdala, and their relevance for anxiety, were studied. CCK/gastrin-like immunoreactive nerve terminals were found to be mainly restricted to the paracapsular intercalated islands and the rostrolateral part of the main intercalated island. Behaviourally, the bilateral microinjection of CCK-4 (0.043-4.3 pmol/side) or CCK-8S (4.3 pmol/side) into the rostrolateral amygdala reduced the open-arm exploration in the elevated plus-maze without affecting locomotion. In contrast, neither CCK-4 nor CCK-8S (0.043-4.3 pmol/side) had any effects in the shock-probe burying test as compared with their saline-treated controls. Biochemically, CCK-4 (0.3 and 1.5 microm), unlike CCK-8S, enhanced significantly the K(+)-stimulated release of [(3)H]GABA from amygdala slices. These effects were fully prevented by prior superfusion of the slices with either the selective CCK-2 receptor antagonist CR2945 (3 microm), or 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), 10 microm, a glutamatergic (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist. It is suggested that CCK modulates glutamate-GABA mechanisms by acting on CCK-2 receptors via volume transmission occurring at the level of the basolateral amygdaloid nucleus and/or by synaptic or perisynaptic volume transmission in the region of the rostrolateral main and paracapsular intercalated islands, resulting in subsequent disinhibition of the central amygdaloid nucleus and anxiety or panic-like behaviour.

  18. Comparison of yttrium and indium complexes of DOTA-BA and DOTA-MBA: models for (90)Y- and (111)In-labeled DOTA-biomolecule conjugates.

    PubMed

    Liu, Shuang; Pietryka, John; Ellars, Charles E; Edwards, D Scott

    2002-01-01

    Yttrium and indium complexes of 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetylbenzylamine (DOTA-BA) and 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetyl-R-(+)-alpha-methylbenzylamine (DOTA-MBA) were prepared in order to study solution structures of (90)Y- and (111)In-labeled DOTA-biomolecule conjugates. (90)Y and (111)In complexes M(L) (M = (90)Y and (111)In; L = DOTA-BA and DOTA-MBA) were prepared from the reaction of MCl(3) with DOTA-BA and DOTA-MBA, respectively, in ammonium acetate buffer. A reverse phase HPLC method revealed that both (90)Y and (111)In complexes show only one radiometric peak in their radio-HPLC chromatograms. It was also found that (111)In(DOTA-BA) and (111)In(DOTA-MBA) are more hydrophilic than their corresponding (90)Y analogues, suggesting different coordination spheres in (111)In and (90)Y complexes of the same DOTA conjugate. Complexes M(L) (M = Y and In; L = DOTA-BA and DOTA-MBA) were prepared and characterized by HPLC, LC-MS, and NMR ((1)H and (13)C) methods. The HPLC concordance experiments for (90)Y(DOTA-MBA)/Y(DOTA-MBA) and (111)In(DOTA-MBA)/In(DOTA-MBA) show that the same complex is prepared at both tracer and macroscopic levels. The NMR data ((1)H and (13)C) clearly demonstrates that Y(DOTA-BA) and Y(DOTA-MBA) exist in solution as one predominant isomer. VT NMR data ((1)H and (13)C) show that In(DOTA-BA) and In(DOTA-MBA) are fluxional at room temperature while Y(DOTA-BA) and Y(DOTA-MBA) become fluxional only at elevated temperatures. The fluxionality of these complexes is due to rapid rotation of acetate/acetamide chelating arms and inversion of ethylenic groups of the macrocyclic ring.

  19. Interferon-induced changes in pharmacokinetics and tumor uptake of 111In-labeled antimelanoma antibody 96. 5 in melanoma patients

    SciTech Connect

    Rosenblum, M.G.; Lamki, L.M.; Murray, J.L.; Carlo, D.J.; Gutterman, J.U.

    1988-04-06

    The type I interferons (both partially purified human leukocyte interferon (HuIFN-alpha) and recombinant alpha interferon) and the type II interferons have been shown to increase the expression of tumor-associated antigens in vitro. To determine whether HuIFN-alpha could increase tumor acquisition of the antimelanoma antibody 96.5 in vivo, five patients with metastatic malignant melanoma were treated with HuIFN-alpha at a dose of 3 X 10(6) units daily by im administration. Twenty-four hours after the first dose of HuIFN-alpha, 1 mg of antibody 96.5 labeled with 5 mCi of /sup 111/In was coadministered with 19 mg of unlabeled 96.5. Five patients matched for metastatic site and lesion size who had not received HuIFN-alpha were also given a dose of 5 mCi of radiolabeled 96.5 at the same total antibody dose (20 mg). In patients treated with HuIFN-alpha, there was a statistically significant increase in the plasma half-life of the /sup 111/In label (39.7 +/- 3.3 hr) compared to the untreated control group (29.8 +/- 3.2 hr). In addition, there was an increase in the apparent volume of distribution of the antibody in the HuIFN-alpha group (5.56 +/- 0.67 L) compared to controls (3.15 +/- 0.5 L) suggesting both an increased immediate extravascular distribution of radiolabeled antibody and a decrease in the subsequent rate of clearance of antibody from plasma. These two phenomena result in a 28% decrease in the area under the concentration curve in the HuIFN-alpha-treated group compared to controls. Computer analysis of whole-body scans from patients showed a threefold increase in radiolabeled antibody distributed to tumor relative to blood pool but no change in organ:blood ratios for liver, spleen, bone, or kidney compared to controls.

  20. Influence of an aliphatic linker between DOTA and synthetic Z(HER2:342) Affibody molecule on targeting properties of the (111)In-labeled conjugate.

    PubMed

    Tolmachev, Vladimir; Feldwisch, Joachim; Lindborg, Malin; Baastrup, Barbro; Sandström, Mattias; Orlova, Anna

    2011-07-01

    Affibody molecules are small (∼6.5 kDa) scaffold proteins suitable for radionuclide imaging of tumor-associated molecular targets. Site-specific labeling of Affibody molecules made by peptide synthesis can be achieved by coupling a chelator to N-terminus in the last synthesis step. The goal of this study was to evaluate the influence of a 6-aminohexanoic linker between DOTA and Z(HER2:342) on targeting properties of (111)In-labeled conjugate. A DOTA-conjugated 6-aminohexanoic linker-containing variant of Z(HER2:342) (ABY-003) was produced by peptide synthesis, and the in vitro binding affinity, specificity and cellular processing were evaluated. The biodistribution of (111)In-ABY-003 in normal mice was compared to (111)In-ABY-002 (DOTA-Z(HER2:342-pep2)) lacking the linker. Tumor-targeting properties of (111)In-ABY-003 were evaluated in mice bearing HER2-expressing xenografts. The dissociation constant of ABY-003 was in the low picomolar range, slightly higher than for ABY-002. (111)In-ABY-003 bound specifically to HER2-expressing cells in vitro. The cellular retention was efficient but slightly worse than for (111)In-ABY-002. In normal mice, the clearance of (111)In-ABY-003 from blood and other tissues was slightly but significantly faster compared to (111)In-ABY-002. Targeting of HER2-expressing xenografts by (111)In-ABY-003 was receptor-specific. Due to faster clearance, the tumor-to-blood ratio for (111)In-ABY-003 at 4 h postinjection was improved compared to (111)In-ABY-002. The capacity of (111)In-ABY-003 to visualize HER2-expressing tumors was confirmed by gamma camera imaging. A 6-aminohexanoic linker between the DOTA chelator and N-terminus of synthetic Z(HER2:342) had a measurable effect on affinity, cellular retention of radioactivity and blood clearance. The linker might be used for modulation of targeting properties of Affibody molecules. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Influence of macrocyclic chelators on the targeting properties of (68)Ga-labeled synthetic affibody molecules: comparison with (111)In-labeled counterparts.

    PubMed

    Strand, Joanna; Honarvar, Hadis; Perols, Anna; Orlova, Anna; Selvaraju, Ram Kumar; Karlström, Amelie Eriksson; Tolmachev, Vladimir

    2013-01-01

    Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide (68)Ga (T1/2=67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule ZHER2:S1 targeting HER2. Affibody molecules were labeled with (68)Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of (68)Ga-DOTA-ZHER2:S1, (68)Ga-NOTA-ZHER2:S1 and (68)Ga-NODAGA-ZHER2:S1, as well as that of their (111)In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for (68)Ga-DOTA-ZHER2:S1 (17.9 ± 0.7%IA/g) was significantly higher than for both (68)Ga-NODAGA-ZHER2:S1 (16.13 ± 0.67%IA/g) and (68)Ga-NOTA-ZHER2:S1 (13 ± 3%IA/g) at 2 h after injection. (68)Ga-NODAGA-ZHER2:S1 had the highest tumor-to-blood ratio (60 ± 10) in comparison with both (68)Ga-DOTA-ZHER2:S1 (28 ± 4) and (68)Ga-NOTA-ZHER2:S1 (42 ± 11). The tumor-to-liver ratio was also higher for (68)Ga-NODAGA-ZHER2:S1 (7 ± 2) than the DOTA and NOTA conjugates (5.5 ± 0.6 vs.3.3 ± 0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for (68)Ga than for (111)In. The results of this study demonstrate that

  2. Optimization of IGF-1R SPECT/CT imaging using 111In-labeled F(ab')2 and Fab fragments of the monoclonal antibody R1507.

    PubMed

    Heskamp, Sandra; van Laarhoven, Hanneke W M; Molkenboer-Kuenen, Janneke D M; Bouwman, Wilbert H; van der Graaf, Winette T A; Oyen, Wim J G; Boerman, Otto C

    2012-08-06

    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-1R antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal antibody directed against IGF-1R. However, antibodies clear slowly from the circulation, resulting in low tumor-to-background ratios early after injection. Therefore, we aimed to accelerate targeting of IGF-1R using radiolabeled R1507 F(ab')2 and Fab fragments. In vitro, immunoreactivity, binding affinity and internalization of R1507 IgG, F(ab')2 and Fab were determined using the triple negative IGF-1R-expressing breast cancer cell line SUM149. In vivo, pharmacokinetics of (111)In-labeled R1507 IgG, F(ab')2 and Fab were studied in mice bearing subcutaneous SUM149 xenografts. SPECT/CT images were acquired and the biodistribution was measured ex vivo. The in vitro binding characteristics of radiolabeled R1507 IgG and F(ab')2 were comparable, whereas the affinity of Fab fragments was significantly lower (Kd: 0.6 nM, 0.7 nM and 3.0 nM for R1507 IgG, F(ab')2 and Fab, respectively). Biodistribution studies showed that the maximum tumor uptake of (111)In-R1507 IgG, F(ab')2 and Fab was 31.8% ID/g (72 h p.i.), 10.0% ID/g (6 h p.i.), and 1.8% ID/g (1 h p.i.), respectively. However, maximal tumor-to-blood ratios for F(ab')2 (24 h p.i.: 7.5) were more than twice as high as those obtained with R1507 (72 h p.i.: 2.8) and Fab (6 h p.i.: 2.8). Injection of an excess of unlabeled R1507 significantly reduced tumor uptake, suggesting that the uptake of R1507 IgG and F(ab')2 was specific for IGF-1R, while the major fraction of the tumor uptake of Fab was nonspecific. IGF-1R-expressing xenografts were visualized with (111)In-F(ab')2 SPECT/CT as early as 6 h p.i., while with R1507 IgG, the tumor could be visualized after 24 h. No specific targeting was observed with (111)In-Fab. (111)In

  3. Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

    PubMed

    Katoh, A; Shoguchi, K; Matsuoka, H; Yoshimura, M; Ohkubo, J-I; Matsuura, T; Maruyama, T; Ishikura, T; Aritomi, T; Fujihara, H; Hashimoto, H; Suzuki, H; Murphy, D; Ueta, Y

    2014-05-01

    The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

  4. Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

    PubMed

    Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts. Copyright © 2014 John Wiley & Sons, Ltd.

  5. Utility of (11)C-methionine and (11)C-donepezil for imaging of Staphylococcus aureus induced osteomyelitis in a juvenile porcine model: comparison to autologous (111)In-labelled leukocytes, (99m) Tc-DPD, and (18)F-FDG.

    PubMed

    Afzelius, Pia; Alstrup, Aage Ko; Schønheyder, Henrik C; Borghammer, Per; Jensen, Svend B; Bender, Dirk; Nielsen, Ole L

    2016-01-01

    The aim of this study was to compare (11)C-methionine and (11)C-donepezil positron emission tomography (PET) with (111)In-labeled leukocyte and (99m) Tc-DPD (Tc-99m 3,3-diphosphono-1,2-propanedicarboxylic acid) single-photon emission computed tomography (SPECT), and (18)F-fluorodeoxyglucose ((18)F-FDG) PET to improve detection of osteomyelitis. The tracers' diagnostic utility where tested in a juvenile porcine hematogenously induced osteomyelitis model comparable to osteomyelitis in children. Five 8-9 weeks old female domestic pigs were scanned seven days after intra-arterial inoculation in the right femoral artery with a porcine strain of Staphylococcus aureus. The sequential scan protocol included Computed Tomography, (11)C-methionine and (11)C-donepezil PET, (99m) Tc-DPD and (111)In-labelled leukocytes scintigraphy, and (18)F-FDG PET. This was followed by necropsy of the pigs and gross pathology, histopathology, and microbial examination. The pigs developed a total of 24 osteomyelitic lesions, 4 lesions characterized as contiguous abscesses and pulmonary abscesses (in two pigs). By comparing the 24 osteomyelitic lesions, (18)F-FDG accumulated in 100%, (111)In-leukocytes in 79%, (11)C-methionine in 79%, (11)C-donepezil in 58%, and (99m) Tc-DPD in none. Overall, (18)F-FDG PET was superior to (111)In-leukocyte SPECT and (11)C-methionine in marking infectious lesions.

  6. Utility of 11C-methionine and 11C-donepezil for imaging of Staphylococcus aureus induced osteomyelitis in a juvenile porcine model: comparison to autologous 111In-labelled leukocytes, 99mTc-DPD, and 18F-FDG

    PubMed Central

    Afzelius, Pia; Alstrup, Aage KO; Schønheyder, Henrik C; Borghammer, Per; Jensen, Svend B; Bender, Dirk; Nielsen, Ole L

    2016-01-01

    The aim of this study was to compare 11C-methionine and 11C-donepezil positron emission tomography (PET) with 111In-labeled leukocyte and 99mTc-DPD (Tc-99m 3,3-diphosphono-1,2-propanedicarboxylic acid) single-photon emission computed tomography (SPECT), and 18F-fluorodeoxyglucose (18F-FDG) PET to improve detection of osteomyelitis. The tracers’ diagnostic utility where tested in a juvenile porcine hematogenously induced osteomyelitis model comparable to osteomyelitis in children. Five 8-9 weeks old female domestic pigs were scanned seven days after intra-arterial inoculation in the right femoral artery with a porcine strain of Staphylococcus aureus. The sequential scan protocol included Computed Tomography, 11C-methionine and 11C-donepezil PET, 99mTc-DPD and 111In-labelled leukocytes scintigraphy, and 18F-FDG PET. This was followed by necropsy of the pigs and gross pathology, histopathology, and microbial examination. The pigs developed a total of 24 osteomyelitic lesions, 4 lesions characterized as contiguous abscesses and pulmonary abscesses (in two pigs). By comparing the 24 osteomyelitic lesions, 18F-FDG accumulated in 100%, 111In-leukocytes in 79%, 11C-methionine in 79%, 11C-donepezil in 58%, and 99mTc-DPD in none. Overall, 18F-FDG PET was superior to 111In-leukocyte SPECT and 11C-methionine in marking infectious lesions. PMID:28078182

  7. 111 In-labeled leukocytes in the detection of prosthetic vascular graft infections

    SciTech Connect

    Williamson, M.R.; Boyd, C.M.; Read, R.C.; Thompson, B.W.; Barnes, R.W.; Shah, H.R.; Balachandran, S.; Ferris, E.J.

    1986-07-01

    Making a clinical diagnosis of infection in prosthetic vascular grafts is difficult but when undiagnosed, this condition has a high mortality rate. Using Indium-111-labeled white-blood cells, 30 scans were performed in 21 patients suspected of having a prosthetic graft infection. The diagnosis of infected graft was confirmed by surgery in all cases, and lack of infection was established by resolution of symptoms with conservative therapy. Twenty-four hour scans of autologous Indium-111 leukocytes were obtained, and correlative CT studies were done in 11 cases. There were 13 infected grafts at surgery (purulent material present), and scans were positive in all (100% sensitivity); of 17 scans, there were 15 true negatives and two false positives (88% specificity). Using the criteria of gas or fluid around the graft, the sensitivity of CT was only 37% in a small subset of these patients. One-half of the cases in which infection was suspected clinically had no infection and had negative scans. Various types of grafts and graft materials were used, and there was no correlation with presence or absence of infection on the basis of the type of graft. Extragraft infection sites were found in five patients. In conclusion, use of Indium-111 leukocytes has been found to be an accurate and valuable diagnostic method for evaluation of suspected prosthetic vascular graft infection, and to have higher diagnostic accuracy than CT.

  8. Size dependent biodistribution and SPECT imaging of (111)In-labeled polymersomes.

    PubMed

    Brinkhuis, René P; Stojanov, Katica; Laverman, Peter; Eilander, Jos; Zuhorn, Inge S; Rutjes, Floris P J T; van Hest, Jan C M

    2012-05-16

    Polymersomes, self-assembled from the block copolymer polybutadiene-block-poly(ethylene glycol), were prepared with well-defined diameters between 90 and 250 nm. The presence of ~1% of diethylene triamine penta acetic acid on the polymersome periphery allowed to chelate radioactive (111)In onto the surface and determine the biodistribution in mice as a function of both the polymersome size and poly(ethylene glycol) corona thickness (i.e., PEG molecular weight). Doubling the PEG molecular weight from 1 kg/mol to 2 kg/mol did not change the blood circulation half-life significantly. However, the size of the different polymersome samples did have a drastic effect on the blood circulation times. It was found that polymersomes of 120 nm and larger become mostly cleared from the blood within 4 h, presumably due to recognition by the reticuloendothelial system. In contrast, smaller polymersomes of around 90 nm circulated much longer. After 24 h more than 30% of the injected dose was still present in the blood pool. This sharp transition in blood circulation kinetics due to size is much more abrupt than observed for liposomes and was additionally visualized by SPECT/CT imaging. These findings should be considered in the formulation and design of polymersomes for biomedical applications. Size, much more than for liposomes, will influence the pharmacokinetics, and therefore, long circulating preparations should be well below 100 nm.

  9. Human biodistribution of sup 111 In-labeled B72. 3 monoclonal antibody

    SciTech Connect

    Harwood, S.J.; Carroll, R.G.; Webster, W.B.; Zangara, L.M.; Laven, D.L.; Morrissey, M.A.; Sinni, B.J. )

    1990-02-01

    The murine IgG1 monoclonal antibody B72.3 reacts with human colorectal, breast, lung, pancreatic, gastric, and ovarian tumors. Human biodistribution studies using intact {sup 131}I-B72.3 have been reported by Carrasquillo et al.. We have performed similar studies on five patients using i.v. infusion of 20 mg of intact {sup 111}In-B72.3 (Cytogen Corp.). Serum clearance is similar with a t1/2 of 64.2 h (range, 44-80) for {sup 111}In-B72.3 and 65 h (range, 32-106) for {sup 131}I-B72.3. However, organ biodistribution is markedly different. For {sup 131}I-B72.3, hepatic and splenic clearance mirrors blood pool clearance. For {sup 111}In-B72.3, there is rapid uptake in tumor, liver, spleen, kidney, lumbar spine, and testes by 2-6 h with no significant clearance over the next 9 days. For {sup 111}In-B72.3, quantitative analysis of liver (from biopsy specimens), spleen, kidney, and lumbar spine (from scintiphoto regions of interest after background subtraction and attenuation correction) shows the following peak organ biodistributions in percentage infused dose: liver, 32%; spleen, 3.9%; kidneys, 3.5%; and lumbar vertebral bodies (marrow sample), 2.7%. For both {sup 111}In-B72.3 and {sup 131}I-B72.3, the principal route of excretion from the body is urinary with excretion rate of {sup 131}I faster than {sup 111}In. The marked differences between {sup 111}In-B72.3 and {sup 131}I-B72.3 biodistribution and clearance strongly influence the dosimetry, immunodetection, and immunotherapeutic potentials of B72.3 MoAb.

  10. Radioimmunodetection of cutaneous T-cell lymphoma with 111In-labeled T101 monoclonal antibody

    SciTech Connect

    Carrasquillo, J.A.; Bunn, P.A. Jr.; Keenan, A.M.; Reynolds, J.C.; Schroff, R.W.; Foon, K.A.; Su, M.H.; Gazdar, A.F.; Mulshine, J.L.; Oldham, R.K.

    1986-09-11

    T101 monoclonal antibody recognizes a pan-T-cell antigen present on normal T cells and also found in high concentrations in cutaneous T-cell lymphoma. We used this antibody, radiolabeled with 111In, in gamma-camera imaging to detect sites of metastatic cutaneous T-cell lymphoma in 11 patients with advanced disease. In all patients, (/sup 111/In)T101 concentrated in pathologically or clinically detected nodes, including those in several previously unsuspected nodal regions. Concentrations (per gram of tissue) ranged from 0.01 to 0.03 percent of the injected dose and were consistently 10 to 100 times higher than previously reported on radioimmunodetection. Focal uptake was seen in skin tumors and heavily infiltrated erythroderma but not in skin plaques. The specificity of tumor targeting was documented by control studies with (/sup 111/In)chloride or (/sup 111/In)9.2.27 (anti-melanoma) monoclonal antibody. Increasing the T101 dose (1 to 50 mg) altered distribution in nontumor tissues. These studies suggest that imaging with (/sup 111/In)T101 may be of value in identifying sites of cutaneous T-cell lymphoma. In contrast to the targeting of solid tumors, the mechanism of localization appears to be related to binding to T cells, which can then carry the radioactivity to involved sites.

  11. Gastrointestinal Hormone Cholecystokinin Increases P-Glycoprotein Membrane Localization and Transport Activity in Caco-2 Cells.

    PubMed

    Yano, Kentaro; Shimizu, Saori; Tomono, Takumi; Ogihara, Takuo

    2017-09-01

    It was reported that stimulation of taste receptor type 2 member 38 by a bitter substance, phenylthiocarbamide (PTC), increased P-glycoprotein (P-gp) mRNA level and transport activity via release of the gastrointestinal hormone cholecystokinin-8 (CCK-8) at 9 h. Therefore, we hypothesized that CCK-8 and PTC might also regulate P-gp activity more rapidly via a different mechanism. As a result, we found that the pretreatment of human colon adenocarcinoma (Caco-2) cells with 10-mM PTC significantly decreased the intracellular accumulation of P-gp substrate rhodamine 123 (Rho123) compared with the control after 90-min incubation. Moreover, CCK-8 treatments significantly reduced the accumulation of Rho123 within 30 min, compared with the control. On the other hand, when Caco-2 cells were pretreated with PTC, the efflux ratio of Rho123 was significantly increased compared with control. The efflux ratio of Rho123 in CCK-8 treatment cells was also significantly increased compared with control. Furthermore, CCK-8 increased the phosphorylation of the scaffold proteins ezrin, radixin, and moesin, which regulate translocation of P-gp to the plasma membrane. Therefore, our results indicate that PTC induced release of CCK-8, which in turn induced the phosphorylation of ezrin, radixin, and moesin proteins, leading to upregulation of P-gp transport activity via increased membrane localization of P-gp. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  12. Radiography, 99mTc-HDP, and 111In labeled vitamin B12 SPECT of canine osteosarcoma: a comparative study.

    PubMed

    Cruz, Robert; Steyn, Phillip; Collins, Douglas; Powers, Barbara; Urigh, John

    2011-01-01

    The objective of this article was to compare radiography, planar bone scintigraphy, and single-photon emission computed tomography (SPECT) to determine the size of osteosarcomas in long bones of dogs. Ten dogs with osteosarcoma in six radii, two humeri, one tibia, and one ulna were evaluated. Macroslides, mediolateral radiographs, planar scintigrams, and sagittal images from SPECT scans were used to obtain measurements. On the scintigraphic images, the edges of the tumor were established using the activity profile imaging tool. The radiographic magnification was factored. The mean percentage of tumor size overestimation was 9.29% on mediolateral radiographs, 5.35% on planar scintigrams, and 33.25% on SPECT images. The correlation coefficient adjusted for sample size was significantly higher (P<0.01) for technetium 99m ((99m)Tc) hydroxyethylene diphosphonate (HDP) (75.5%) and radiography (61.3%) compared with indium 111-vitamin B(12) (28.3%). The correlation coefficient for (99m)Tc-HDP was higher than that obtained for radiographs; however, statistical difference between the two variables was not demonstrated (P>0.05). (99m)Tc bone scan is a good estimator of intramedullary size of osteosarcoma in long bones when the activity profile tool to determine the margin of the tumor is used.

  13. Anticholecystokinin activities of loxiglumide.

    PubMed

    Setnikar, I; Bani, M; Cereda, R; Chisté, R; Makovec, F; Pacini, M A; Revel, L

    1987-10-01

    The anticholecystokinin activities of loxiglumide, (D,L-4-(3,4-dichloro-benzoylamino)-5-(N-3-methoxypropyl-pentylamino++ +)-5-oxo- pentanoic acid, CR 1505) are described. Loxiglumide antagonizes in vivo the contractions of the gall bladder of guinea pig induced or mediated by cholecystokinin-8 (CCK-8) (i.v. ED50 = 0.24 mumol/kg), the emptying of the gall bladder of the mouse induced by CCK-8 (i.v. ED50 = 29 mumol/kg, oral ED50 = 42 mumol/kg), the retardation of gastric emptying of the rat induced by CCK-8 (i.p. ED50 = 13 mumol/kg), the retardation of the pyloric transit in the mouse induced by CCK-8 (i.v. ED50 = 3.7 mumol/kg, oral ED50 = 11 mumol/kg), the hypermotility of the ileum of the rabbit induced by CCK-8 (i.v. ED50 = 1.2 mumol/kg) and the contractions of the gall bladder of the non-anesthetized dog induced by caerulein (i.v. ED50 ca. 11 mumol/kg). Loxiglumide also antagonizes the satiety behaviour of the rat elicited by CCK-8 (i.p. ED50 = 0.65 mumol/kg) and the exocrine pancreatic hypersecretion in the anaesthetized dog induced by CCK-8 (i.v. ED50 ca. 0.35 mumol/kg). Loxiglumide has a simple, non-polypeptidic chemical structure and is active after parenteral and after oral administration.

  14. Quantification of the Sulfated Cholecystokinin CCK8 in Hamster Plasma Using Immunoprecipitation-Liquid Chromatography-Mass Spectrometry/Mass Spectrometry

    USDA-ARS?s Scientific Manuscript database

    Cholecystokinin (CCK) and the different molecular forms of CCK are well established as biomarkers for satiety. CCK hormone and the different biologically active and inactive molecular forms have been shown to influence food intake associated with satiety and are predominately secreted from the gut....

  15. Autoradiographical detection of cholecystokinin-A receptors in primate brain using sup 125 I-Bolton Hunter CCK-8 and 3H-MK-329

    SciTech Connect

    Hill, D.R.; Shaw, T.M.; Graham, W.; Woodruff, G.N. )

    1990-04-01

    In vitro autoradiography was performed in order to visualize cholecystokinin-A (CCK-A) receptors in sections of Cynomolgus monkey brain. CCK-A receptors were defined as those which displayed high affinity for the selective non-peptide antagonist MK-329 (L-364,718) and were detected in several regions by selective inhibition of 125I-Bolton Hunter CCK using MK-329 or direct labeling with 3H-MK-329. In the caudal medulla, high densities of CCK-A sites were present in the nucleus tractus solitarius, especially the caudal and medial aspects, and also the dorsal motor nucleus of the vagus. CCK-A sites were localized to a number of hypothalamic nuclei such as the supraoptic and paraventricular nuclei, the dorsomedial and infundibular nuclei as well as the neurohypophysis. The mammillary bodies and supramammillary nuclei also contained CCK-A receptor sites. High concentrations of CCK-A receptors were present in the substantia nigra zona compacta and also the ventral tegmental area and may be associated with dopamine cell bodies. Binding of 3H-MK-329 was also detected in parts of the caudate nucleus and ventral putamen. The detection, by autoradiographical means, of CCK-A receptors throughout the Cynomolgus monkey brain contrasts with similar studies performed using rodents and suggests differences in the density and, perhaps, the importance of CCK-A receptors in the primate as opposed to the rodent. The data suggest the possibility that CCK-A receptors may be involved in a number of important brain functions as diverse as the processing of sensory information from the gut, the regulation of hormone secretion, and the activity of dopamine cell activity.

  16. Molecular strategies and 111in-labelled somatostatin analogues in defining the management of neuroendocrine tumour disease: a new paradigm for surgical management.

    PubMed

    Modlin, I M; Kidd, M; Hinoue, T; Lye, K D; Murren, J; Argiris, A

    2003-06-01

    This manuscript provides a gene-chip examination of gastric ECL cell proliferation in an animal model of neuroendocrine tumour disease. Data that were used to identify molecular targets were then utilised to develop novel therapeutic strategies as appropriate adjuncts to surgery in human disease. Alterations in growth-mediated cell signaling (the AP-1 pathway) and in the cell cycle were identified in ECL cell tumours in the animal model and confirmed in human tumour tissue. The growth-inhibitory somatostatin receptor subtype 2 was identified as a potential clinical target. An investigation of patients with neuroendocrine tumours treated using SSTR2 targeted radiotherapy [111In]pentetreotide producing encouraging preliminary results. Fifty-six per cent of patients with evaluable hormone markers demonstrated stable levels or a significant decrease in one or more measured markers. This data demonstrate that gene pathways recognised to be altered in an animal model of a human disease can be used to identify therapeutic agents. This approach was successfully used to discover novel strategies that can be both effective and appropriate adjuncts to surgery for patients with neuroendocrine tumour disease.

  17. Uptake of 111In-labeled fully human monoclonal antibody TSP-A18 reflects transferrin receptor expression in normal organs and tissues of mice.

    PubMed

    Sugyo, Aya; Tsuji, Atsushi B; Sudo, Hitomi; Nomura, Fumiko; Satoh, Hirokazu; Koizumi, Mitsuru; Kurosawa, Gene; Kurosawa, Yoshikazu; Saga, Tsuneo

    2017-03-01

    Transferrin receptor (TfR) is an attractive molecule for targeted therapy of cancer. Various TfR-targeted therapeutic agents such as anti-TfR antibodies conjugated with anticancer agents have been developed. An antibody that recognizes both human and murine TfR is needed to predict the toxicity of antibody-based agents before clinical trials, there is no such antibody to date. In this study, a new fully human monoclonal antibody TSP-A18 that recognizes both human and murine TfR was developed and the correlation analysis of the radiolabeled antibody uptake and TfR expression in two murine strains was conducted. TSP-A18 was selected using extracellular portions of human and murine TfR from a human antibody library. The cross-reactivity of TSP-A18 with human and murine cells was confirmed by flow cytometry. Cell binding and competitive inhibition assays with [111In]TSP-A18 showed that TSP-A18 bound highly to TfR-expressing MIAPaCa-2 cells with high affinity. Biodistribution studies of [111In]TSP-A18 and [67Ga]citrate (a transferrin-mediated imaging probe) were conducted in C57BL/6J and BALB/c-nu/nu mice. [111In]TSP-A18 was accumulated highly in the spleen and bone containing marrow component of both strains, whereas high [67Ga]citrate uptake was only observed in bone containing marrow component and not in the spleen. Western blotting indicated the spleen showed the strongest TfR expression compared with other organs in both strains. There was significant correlation between [111In]TSP-A18 uptake and TfR protein expression in both strains, whereas there was significant correlation of [67Ga]citrate uptake with TfR expression only in C57BL/6J. These findings suggest that the difference in TfR expression between murine strains should be carefully considered when testing for the toxicity of anti-TfR antibody in mice and the uptake of anti-TfR antibody could reflect tissue TfR expression more accurately compared with that of transferrin-mediated imaging probe such as [67Ga]citrate.

  18. Development of (111)In-labeled exendin(9-39) derivatives for single-photon emission computed tomography imaging of insulinoma.

    PubMed

    Kimura, Hiroyuki; Matsuda, Hirokazu; Ogawa, Yu; Fujimoto, Hiroyuki; Toyoda, Kentaro; Fujita, Naotaka; Arimitsu, Kenji; Hamamatsu, Keita; Yagi, Yusuke; Ono, Masahiro; Inagaki, Nobuya; Saji, Hideo

    2017-02-15

    Insulinoma is a tumor derived from pancreatic β-cells, and the resulting hyperinsulinemia leads to characteristic hypoglycemia. Recent studies have reported the frequent overexpression of glucagon-like peptide-1 receptor (GLP-1R) in human insulinomas, suggesting that the binding of a radiolabeled compound to GLP-1R is useful for the imaging of such tumors. Exendin(9-39), a fragment peptide of exendin-3 and -4, binds GLP-1R with high affinity and acts as an antagonist. Accordingly, radiolabeled exendin(9-39) derivatives have also been investigated as insulinoma imaging probes that might be less likely to induce hypoglycemia. In this study, we synthesized a novel indium-111 ((111)In)-benzyl-diethylenetriaminepentaacetic acid ((111)In-BnDTPA)-conjugated exendin(9-39), (111)In-BnDTPA-exendin(9-39), and evaluated its utility as a probe for the SPECT imaging of insulinoma. natIn-BnDTPA-exendin(9-39) exhibited a high affinity for GLP-1R (IC50=2.5nM), stability in plasma, and a specific activity that improved following reactions with a solvent and solubilizer. Regarding the in vivo biodistribution of (111)In-BnDTPA-exendin(9-39) in INS-1 tumor-bearing mice, high uptake levels were observed in tumors (14.6%ID/g at 15min), with corresponding high tumor-to-blood (T/B), tumor-to-muscle (T/M), and tumor-to-pancreas (T/P) ratios (T/B=2.55, T/M=22.7, T/P=2.7 at 1h). The pre-administration of excess nonradioactive exendin(9-39) significantly reduced accumulation in both the tumor and pancreas (76% and 68% inhibition, respectively) at 1h after (111)In-BnDTPA-exendin(9-39) injection, indicating that the GLP-1R mediated a majority of (111)In-BnDTPA-exendin(9-39) uptake in the tumor and pancreas. Finally, (111)In-BnDTPA-exendin(9-39) SPECT/CT studies in mice yielded clear images of tumors at 30min post-injection. These results suggest that (111)In-BnDTPA-exendin(9-39) could be a useful SPECT molecular imaging probe for the detection and exact localization of insulinomas. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Protein kinase D1 mediates NF-κB activation induced by cholecystokinin and cholinergic signaling in pancreatic acinar cells

    PubMed Central

    Yuan, Jingzhen; Lugea, Aurelia; Zheng, Ling; Gukovsky, Ilya; Edderkaoui, Mouad; Rozengurt, Enrique; Pandol, Stephen J.

    2008-01-01

    The transcription factor NF-κB plays a critical role in inflammatory and cell death responses during acute pancreatitis. Previous studies in our laboratory demonstrated that protein kinase C (PKC) isoforms PKCδ and ɛ are key regulators of NF-κB activation induced by cholecystokinin-8 (CCK-8), tumor necrosis factor-α, and ethanol. However, the downstream participants in regulating NF-κB activation in exocrine pancreas remain poorly understood. Here, we demonstrate that protein kinase D1 (PKD1) is a key downstream target of PKCδ and PKCɛ in pancreatic acinar cells stimulated by two major secretagogues, CCK-8 and the cholinergic agonist carbachol (CCh), and that PKD1 is necessary for NF-κB activation induced by CCK-8 and CCh. Both CCK-8 and CCh dose dependently induced a rapid and striking activation of PKD1 in rat pancreatic acinar cells, as measured by in vitro kinase assay and by phosphorylation at PKD1 activation loop (Ser744/748) or autophosphorylation site (Ser916). The phosphorylation and activation of PKD1 correlated with NF-κB activity stimulated by CCK-8 or CCh, as measured by NF-κB DNA binding. Either inhibition of PKCδ or ɛ by isoform-specific inhibitory peptides, genetic deletion of PKCδ and ɛ in pancreatic acinar cells, or knockdown of PKD1 by using small interfering RNAs in AR42J cells resulted in a marked decrease in PKD1 and NF-κB activation stimulated by CCK-8 or CCh. Conversely, overexpression of PKD1 resulted in augmentation of CCK-8- and CCh-stimulated NF-κB activation. Finally, the kinetics of PKD1 and NF-κB activation during cerulein-induced rat pancreatitis showed that both PKD1 and NF-κB activation were early events during acute pancreatitis and that their time courses of response were similar. Our results identify PKD1 as a novel early convergent point for PKCδ and ɛ in the signaling pathways mediating NF-κB activation in pancreatitis. PMID:18845574

  20. Pancreas-specific aquaporin 12 null mice showed increased susceptibility to caerulein-induced acute pancreatitis.

    PubMed

    Ohta, Eriko; Itoh, Tomohiro; Nemoto, Tomomi; Kumagai, Jiro; Ko, Shigeru B H; Ishibashi, Kenichi; Ohno, Mayuko; Uchida, Keiko; Ohta, Akihito; Sohara, Eisei; Uchida, Shinichi; Sasaki, Sei; Rai, Tatemitsu

    2009-12-01

    Aquaporin 12 (AQP12) is the most recently identified member of the mammalian AQP family and is specifically expressed in pancreatic acinar cells. In vitro expression studies have revealed that AQP12 is localized at intracellular sites. To determine the physiological roles of AQP12 in the pancreas, we generated knockout mice for this gene (AQP12-KO). No obvious differences were observed under normal conditions between wild-type (WT) and AQP12-KO mice in terms of growth, blood chemistry, pancreatic fluid content, or histology. However, when we induced pancreatitis through the administration of a cholecystokinin-8 (CCK-8) analog, the AQP12-KO mice showed more severe pathological damage to this organ than WT mice. Furthermore, when we analyzed exocytosis in the pancreatic acini using a two-photon excitation imaging method, the results revealed larger exocytotic vesicles (vacuoles) in the acini of AQP12-KO mice at a high CCK-8 dose (100 nM). From these results, we conclude that AQP12 may function in the mechanisms that control the proper secretion of pancreatic fluid following rapid and intense stimulation.

  1. Existence of serotonin and neuropeptides-immunoreactive endocrine cells in the small and large intestines of the mole-rats (Spalax leucodon).

    PubMed

    Yaman, M; Bayrakdar, A; Tarakçı, B G

    2012-08-01

    The present study was conducted to clarify the regional distribution and relative frequency of endocrine cells secreting serotonin, substance P (SP), cholecystokinin-8 (CCK-8), vasoactive intestinal polypeptide (VIP) and neurotensin in the small and large intestine of the mole-rats (Spalax leucodon), by specific immunohistochemical methods. In the small and large intestine of mole-rats (Spalax leucodon), serotonin, SP and VIP were identified with various frequencies, but CCK-8 and neurotensin were not observed. Most of the IR cells in the small and large intestine were located in the intestinal crypt and epithelium however, they were more frequency in the intestinal crypt. Serotonin-IR cells were detected throughout the whole intestinal tract, predominantly in the duodenum and colon. SP-IR cells were demonstrated throughout the whole intestinal tract except for the ileum and rectum with highest frequencies in the cecum. VIP-IR cells were found in all parts of the small intestine except for the large intestine. In conclusion, the general distribution patterns and relative frequency of intestinal endocrine cells of the mole-rats (Spalax leucodon) was similar to those of some rodent species. However, some species-dependent unique distributions and frequencies characteristics of endocrine cells were also observed in the present study.

  2. Endocrine cells producing peptide hormones in the intestine of Nile tilapia: distribution and effects of feeding and fasting on the cell density.

    PubMed

    Pereira, Raquel Tatiane; de Freitas, Thaiza Rodrigues; de Oliveira, Izabela Regina Cardoso; Costa, Leandro Santos; Vigliano, Fabricio Andrés; Rosa, Priscila Vieira

    2017-05-13

    Endocrine cells (ECs) act as a luminal surveillance system responding to either the presence or absence of food in the gut through the secretion of peptide hormones. The aim of this study was to analyze the effects of feeding and fasting on the EC peptide-specific distribution along the intestine of Nile tilapia. We assessed the density of ECs producing gastrin (GAS), cholecystokinin-8 (CCK-8), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) in nine segments of the intestine using immunohistochemistry. Our results show that ECs immunoreactive to CCK-8, GAS, NPY, and CGRP can be found along all the intestinal segments sampled, from the midgut to hindgut, although differences in their distribution along the gut were observed. Regarding nutrient status, we found that the anterior segments of the midgut seem to be the main site responding to luminal changes in Nile tilapia. The NPY+ and CGRP+ EC densities increased in the fasted group, while the amount of CCK-8+ ECs were higher in the fed group. No effects of fasting or feeding were found in the GAS+ EC densities. Changes in ECs density were found only at the anterior segments of the intestine which may be due to the correlation between vagus nerve anatomy, EC location, and peptide turnover. Lastly, ECs may need to be considered an active cell subpopulation that may adapt and respond to different nutrient status as stimuli. Due to the complexity of the enteroendocrine system and its importance in fish nutrition, much remains to be elucidated and it deserves closer attention.

  3. Biodistribution, Pharmacokinetics, and Dosimetry of (177)Lu-, (90)Y-, and (111)In-Labeled Somatostatin Receptor Antagonist OPS201 in Comparison to the Agonist (177)Lu-DOTATATE: The Mass Effect.

    PubMed

    Nicolas, Guillaume P; Mansi, Rosalba; McDougall, Lisa; Kaufmann, Jens; Bouterfa, Hakim; Wild, Damian; Fani, Melpomeni

    2017-09-01

    Radiolabeled somatostatin receptor (SSTR) antagonists have shown in vivo higher uptake in SSTR-expressing tumors than agonists. In this preclinical study, the SSTR2 antagonist OPS201 (DOTA-JR11; DOTA-[Cpa-c(DCys-Aph(Hor)-DAph(Cbm)-Lys-Thr-Cys)-DTyr-NH2]) labeled with (177)Lu, (90)Y, and (111)In was compared with the SSTR2 agonist (177)Lu-DOTATATE. Methods: Biodistribution, pharmacokinetics, SPECT/CT, and dosimetry studies were performed to assess the bioequivalence of all radiotracers. Use of escalated peptide mass and nephroprotective agents were systematically investigated. Results: The tumor residence time was 15.6 h (13.4-17.7) for (177)Lu-OPS201 (10 pmol) and 6.4 h (5.4-7.3) for (177)Lu-DOTATATE, resulting in a 2.5-times-higher tumor dose for the antagonist than for the agonist (0.854 vs. 0.333 mGy/MBq for a 4-cm tumor). The overall tumor-to-kidney dose ratio was approximately 24% and 32% higher for (177)Lu-OPS201 than for (90)Y-OPS201 and (177)Lu-DOTATATE, respectively. (111)In-OPS201 had a biodistribution significantly different from (90)Y-OPS201 and is therefore not a surrogate for (90)Y-OPS201 dosimetry studies. Importantly, and in contrast to (177)Lu-DOTATATE, injection of 10, 200, and 2,000 pmol of (177)Lu-OPS201 did not cause any relevant tumor saturation, with tumor uptake 4 h after injection: 23.9, 24.9, and 18.8 percentage of injected activity per gram of tissue (%IA/g), respectively, for the antagonist (P > 0.05), as compared with 17.8, 12.0, and 9.9 %IA/g for the agonist (P < 0.05). Increasing the peptide mass of (177)Lu-OPS201 from 10 to 200 pmol drastically decreased the effective dose from 0.0908 to 0.0184 mSv/MBq and decreased the uptake in the liver, bone marrow, and all SSTR2-expressing organs; thus, the therapeutic index improved considerably. Lysine and succinylated gelatine, alone or in combination, significantly reduced the renal dose of (177)Lu-OPS201 compared with the control group, by 45%, 25%, and 40%, respectively (P < 0.05). The reduction was similar for 10 and 200 pmol, whereas lysine performed better than succinylated gelatine. Conclusion:(177)Lu-OPS201 exhibits higher tumor uptake, longer tumor residence time, and improved tumor-to-kidney dose ratio compared with (177)Lu-DOTATATE and (90)Y-OPS201. Importantly, the mass-escalation study indicates that an optimized antagonist mass might further improve the safety window of peptide receptor radionuclide therapy by reducing the liver and bone marrow doses as well as the effective dose. Clinical studies are warranted to confirm the efficacy and advantageous toxicity profile of (177)Lu-OPS201. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

  4. A Monte Carlo approach to small-scale dosimetry of solid tumour microvasculature for nuclear medicine therapies with (223)Ra-, (131)I-, (177)Lu- and (111)In-labelled radiopharmaceuticals.

    PubMed

    Amato, Ernesto; Leotta, Salvatore; Italiano, Antonio; Baldari, Sergio

    2015-07-01

    The small-scale dosimetry of radionuclides in solid-tumours is directly related to the intra-tumoral distribution of the administered radiopharmaceutical, which is affected by its egress from the vasculature and dispersion within the tumour. The aim of the present study was to evaluate the combined dosimetric effects of radiopharmaceutical distribution and range of the emitted radiation in a model of tumour microvasculature. We developed a computational model of solid-tumour microenvironment around a blood capillary vessel, and we simulated the transport of radiation emitted by (223)Ra, (111)In, (131)I and (177)Lu using the GEANT4 Monte Carlo. For each nuclide, several models of radiopharmaceutical dispersion throughout the capillary vessel were considered. Radial dose profiles around the capillary vessel, the Initial Radioactivity (IR) necessary to deposit 100 Gy of dose at the edge of the viable tumour-cell region, the Endothelial Cell Mean Dose (ECMD) and the Tumour Edge Mean Dose (TEMD), i.e. the mean dose imparted at the 250-μm layer of tissue, were computed. The results for beta and Auger emitters demonstrate that the photon dose is about three to four orders of magnitude lower than that deposited by electrons. For (223)Ra, the beta emissions of its progeny deliver a dose about three orders of magnitude lower than that delivered by the alpha emissions. Such results may help to characterize the dose inhomogeneities in solid tumour therapies with radiopharmaceuticals, taking into account the interplay between drug distribution from vasculature and range of ionizing radiations.

  5. (111)In-labeled trastuzumab-modified gold nanoparticles are cytotoxic in vitro to HER2-positive breast cancer cells and arrest tumor growth in vivo in athymic mice after intratumoral injection.

    PubMed

    Cai, Zhongli; Chattopadhyay, Niladri; Yang, Kaiyu; Kwon, Yongkyu Luke; Yook, Simmyung; Pignol, Jean-Philippe; Reilly, Raymond M

    2016-12-01

    Gold nanoparticles (AuNP; 30nm) were modified with polyethylene glycol (PEG) chains linked to trastuzumab for binding to HER2-positive breast cancer (BC) cells and diethylenetriaminepentaacetic acid (DTPA) for complexing the Auger electron-emitter, (111)In (trastuzumab-AuNP-(111)In). Our objective was to determine the cytotoxicity of trastuzumab-AuNP-(111)In on HER2-positive BC cells in vitro and evaluate its tumor growth inhibition properties and normal tissue toxicity in vivo following intratumoral (i.t.) injection in mice with s.c. HER2-overexpressing BC xenografts. Binding and internalization of trastuzumab-AuNP-(111)In or non-targeted AuNP-(111)In in SK-BR-3 (1-2×10(6) HER2/cell) and MDA-MB-361 (5×10(5) HER2/cell) human BC cells were studied. The surviving fraction (SF) of SK-BR-3 or MDA-MB-361 cells exposed to trastuzumab-AuNP-(111)In or AuNP-(111)In was determined. DNA double-strand breaks (DSBs) were assayed by probing for γ-H2AX. Tumor growth was monitored over 70days in CD1 athymic mice with s.c. MDA-MB-361 xenografts after i.t. injection of 10MBq (0.7mg; 2.6×10(12) AuNP) of trastuzumab-AuNP-(111)In and normal tissue toxicity was assessed by monitoring body weight, complete blood cell (CBC) counts and serum alanine aminotransferase (ALT) and creatinine (Cr). Trastuzumab-AuNP-(111)In was specifically bound by SK-BR-3 and MDA-MB-361 cells. Trastuzumab-AuNP-(111)In was more efficiently internalized than AuNP-(111)In and localized to a peri-nuclear region. The SF fraction of SK-BR-3 cells was reduced by 1.8-fold by treatment with 3nM (7MBq/mL) of trastuzumab-AuNP-(111)In. The SF of MDA-MB-361 cells was reduced by 3.7-fold at 14.4nM (33.6MBq/mL). In comparison, non-targeted AuNP-(111)In at these concentrations reduced the SF of SK-BR-3 or MDA-MB-361 cells by 1.2-fold (P=0.03) and 1.7-fold (P<0.0001), respectively. DNA DSBs were greater in SK-BR-3 and MDA-MB-361 cells exposed to trastuzumab-AuNP-(111)In compared to AuNP-(111)In, but unlabeled trastuzumab-AuNP did not increase DNA DSBs. Local i.t. injection of trastuzumab-AuNP-(111)In in CD1 athymic mice with s.c. MDA-MB-361 tumors arrested tumor growth for 70days. There was no apparent normal tissue toxicity. The radiation absorbed dose deposited in the tumor by trastuzumab-AuNP-(111)In was 60.5Gy, while normal organs received <0.9Gy. These results are promising for further development of trastuzumab-AuNP-(111)In as a novel Auger electron-emitting radiation nanomedicine for local treatment of HER2-positive BC. A local radiation treatment for HER2-positive BC based on AuNP modified with trastuzumab and labeled with the Auger electron-emitter, (111)In was developed and shown to arrest tumor growth with no normal tissue toxicity. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Imaging agents for in vivo molecular profiling of disseminated prostate cancer--targeting EGFR receptors in prostate cancer: comparison of cellular processing of [111In]-labeled affibody molecule Z(EGFR:2377) and cetuximab.

    PubMed

    Malmberg, Jennie; Tolmachev, Vladimir; Orlova, Anna

    2011-04-01

    Expression of receptor tyrosine-kinase (RTK) EGFR is low in normal prostate, but increases in prostate cancer. This receptor is significantly up-regulated as tumors progress into higher grade, androgen-insensitive and metastatic lesions. The up-regulated receptors could serve as targets for novel selective anti-cancer drugs, e.g. antibodies and tyrosine kinase inhibitors. Radionuclide imaging of RTK can facilitate patient stratification and monitoring of anti-RTK therapy of prostate cancer. The goal of the study was to evaluate binding and cellar processing of radiolabeled EGFR-targeting conjugates by prostate cancer cell lines. Receptor expression of EGFR was studied in three prostate cancer cell lines: DU145 (brain metastasis of PC, hormone insensitive), PC3 (bone metastasis of PC) and LNCaP (lymph node metastasis of PC, androgen and estrogen receptor positive). Uptake and internalization of anti-EGFR mAbs (cetuximab) and affibody molecule (Z2377) labeled with indium-111 was investigated. EGFR expression on prostate cancer cell lines was clearly demonstrated. Both labelled conjugates 111In-Z2377 and 111In-cetuximab bound to prostate cancer cells in the receptor mediated model. Expression levels were modest but correlate with degree of hormone independence. Internalization of Affibody molecules was relatively slow in all cell lines. Internalization of mAbs was more rapid. The level of EGFR expression in these cell lines is sufficient for in vivo molecular imaging. Slow internalization indicates possibility of the use of non-residualizing labels for affibody molecules.

  7. Neurohumoral regulation of body weight gain.

    PubMed

    Devaskar, S U

    2001-09-01

    The regulation of body weight is a complex process which relies on a balance between supply of nutrients and demand on these nutrients in the form of energy expenditure. Various central and peripheral mechanisms play a crucial role in maintaining this balance. While various neuropeptides in the central nervous system (CNS), particularly in the hypothalamus, maintain the necessary harmony between hyperphagia and anorexia, peripheral signals arising from the gastrointestinal tract (cholecystokinin-8 [CCK-8], amylin), pancreas (insulin) and adipose tissue (leptin) provide the necessary stimuli or a feedback inhibition for the synthesis and secretion of these hypothalamic neuropeptides. Various metabolites of the carbohydrate and fat metabolism are also involved in regulating the neuronal activity in the hypothalamus which ultimately leads to a release of key neuropeptides. In addition to the central mechanisms, peripheral mechanisms that regulate energy expenditure, particularly in the brown adipose tissue and skeletal muscle, are critical in maintaining the overall balance. Insight into these mechanisms sets the stage for developing novel strategies in the treatment of emerging childhood diseases such as obesity, anorexia nervosa, and bulimia. Further, delineation of these processes in the fetus and newborn sets the stage for investigating their role in molding the adult phenotype due to intrauterine adaptations.

  8. Biodistributions of 177Lu- and 111In- labeled 7E11 Antibodies to Prostate-Specific Membrane Antigen in Xenograft Model of Prostate Cancer and Potential Use of 111In-7E11 as a Pretherapeutic Agent for 177Lu-7E11 Radioimmunotherapy

    PubMed Central

    Pan, Mei-Hsiu; Gao, Dong-Wei; Feng, Jinjin; He, Jiang; Seo, Youngho; Tedesco, John; Wolodzko, John G.; Hasegawa, Bruce H.; Franc, Benjamin L.

    2010-01-01

    Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein highly expressed in many prostate cancers, and can be targeted with radiolabeled antibodies for diagnosis and treatment of this disease. To serve as a radioimmunotherapeutic agent, a kinetically inert conjugate is desired to maximize tumor uptake and tumor radiation dose with minimal nonspecific exposure to bone marrow and other major organs. In this study, we assessed the pharmacokinetics and biodistribution of the 7E11 monoclonal antibody (MAb) radiolabeled with the lutetium-177 (177Lu) - tetraazacyclododecanetetraacetic acid (DOTA) conjugate system (177Lu-7E11) versus those of the 7E11 MAb radiolabeled with the indium-111 (111In) – glycyl-tyrosyl-(N,-diethylenetriaminepentaacetic acid)-lysine hydrochloride (DTPA) conjugate system (111In-7E11, also known as ProstaScint®), to determine the feasibility of using 111In-7E11 as a pretherapeutic agent for 177Lu-7E11 radioimmunotherapy. Pharmacokinetic and biodistribution studies of 177Lu-7E11 in LNCaP xenograft mice were performed at 2, 8, 12, 24, 72, and 168 hours after radiopharmaceutical administration. For 111In-7E11, pharmacokinetic and biodistribution studies were performed at 8, 24, and 72 hours. Parallel studies of 177Lu-7E11 in nontumor bearing mice at 8, 24, and 72 hours postinjection served as controls. Gamma scintigraphy was performed, followed by autoradiography and tissue counting to demonstrate and quantify the distributions of radioconjugated MAb in the tumor and normal tissues. Both 177Lu- and 111In- 7E11 conjugates demonstrated an early blood pool phase in which uptake was dominated by the blood, lung, spleen and liver, followed by uptake and retention of the radiolabeled antibody in the tumor which was most prominent at 24 h. Total accumulation of radioconjugated MAb in tumor at 24 h was greater in the case of 177Lu-7E11 in comparison to that of 111In-7E11. Continued accumulation in tumor was observed for the entire time course studied for both 177Lu-7E11 and 111In-7E11. The liver was the only major organ demonstrating a significant difference in accumulation between the two conjugates. In conclusion, pharmacokinetic and biodistribution studies of 177Lu-7E11 in LNCaP xenograft mouse models support its potential application as a radioimmunotherapeutic agent targeting prostate cancer, and the distribution and tumor uptake of 111In-7E11 appear to be similar to those of 177Lu-7E11, supporting its use as a pretherapeutic tool to assess the potential accumulation of 177Lu-7E11 radioimmunotherapeutic at sites of prostate cancer. However, the different accumulation patterns of the 111In and 177 Lu immunoconjugates in liver will likely prevent the use of 111In-7E11 as a true dosimetry tool for 177Lu-7E11 radioimmunotherapy. PMID:19034582

  9. Sedative action of cholecystokinin octapeptide on behavioral excitation by thyrotropin releasing hormone and methamphetamine in the rat.

    PubMed

    Katsuura, G; Itoh, S

    1982-01-01

    Intracerebroventricular (i.c.v.) injection of C-terminal octapeptide of cholecystokinin (CCK-8) in rats prolonged pentobarbital- and ethanol-induced sleeping time, but non-sulfated CCD-8 (CCK-8-NS) had no effect and caerulein showed a tendency to prolong the pentobarbital narcosis. On the other hand, i.c.v. injection of thyrotropin releasing hormone (TRH) shortened the sleeping time and the effect of CCK-8 was apparently antagonized by combined administration of TRH. Spontaneous locomotor activity in the late morning and early afternoon was not affected by CCK-8, but it increased following i.c.v. injection of CCK-8-NS. Hyperactivity produced by TRH and methamphetamine was suppressed by i.c.v. injection of CCK-8, while CCK-8-NS showed a tendency to enhance the methamphetamine-induced hyperactivity and caerulein had no effect. These results indicate that CCK-8 has a sedative action and antagonizes the behavioral excitation caused by TRH and methamphetamine, but that the effects of CCK-8-NS and caerulein were rather the opposite of those of CCK-8. In an additional experiment the TRH-induced body shaking response was not affected by combined administration of CCK-8.

  10. Effects of phorbol ester on cholecystokinin octapeptide-evoked exocrine pancreatic secretion in the rat.

    PubMed Central

    Francis, L P; Camello, P J; Singh, J; Salido, G M; Madrid, J A

    1990-01-01

    1. A comparative study was made of the effect of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) on cholecystokinin octapeptide-evoked exocrine pancreatic secretion in the anaesthetized rat and isolated permeabilized pancreatic acinar cells. 2. Cholecystokinin octapeptide (CCK8; 0.10-6.40 nmol (kg body weight)-1) induced dose-dependent increases in pancreatic juice flow, total protein output and amylase release in the anaesthetized rat. 3. Administration of TPA (10(-8) mol (kg body weight)-1) in combination with CCK8 resulted in marked attenuation of the CCK8-evoked secretory response. 4. Simultaneous injection of polymyxin B (10(-8) mol (kg body weight)-1), an inhibitor of protein kinase C, with TPA and CCK8 reversed the inhibitory effect of the phorbol ester on CCK8-induced pancreatic juice flow, total protein output and amylase release. 5. In permeabilized rat pancreatic acini CCK8 (10(-13)-10(-9) M) elicited dose-dependent increases in [3H]leucine-labelled protein secretion (3H-labelled protein release). Combining TPA (10(-8) M) with CCK8 resulted in an inhibition of the CCK8-induced 3H-labelled protein release especially at lower concentrations of CCK8. At higher concentrations of CCK8, TPA was unable to inhibit the CCK8-evoked 3H-labelled protein release. Again, polymyxin B reversed the TPA-induced inhibition of CCK8-evoked 3H-labelled protein output. 6. The results indicate that protein kinase C activation may play an important physiological role in modulating the CCK8-evoked secretory response in rat pancreas in vivo and in vitro. PMID:1712842

  11. Effects of Exogenous Cholecystokinin Octapeptide on Acquisition of Naloxone Precipitated Withdrawal Induced Conditioned Place Aversion in Rats

    PubMed Central

    Ma, Chunling; Meng, Yanxin; Li, Shujin; Ni, Zhiyu; Cong, Bin

    2012-01-01

    Cholecystokinin octapeptide (CCK-8), a gut-brain peptide, regulates a variety of physiological behavioral processes. Previously, we reported that exogenous CCK-8 attenuated morphine-induced conditioned place preference, but the possible effects of CCK-8 on aversively motivated drug seeking remained unclear. To investigate the effects of endogenous and exogenous CCK on negative components of morphine withdrawal, we evaluated the effects of CCK receptor antagonists and CCK-8 on the naloxone-precipitated withdrawal-induced conditioned place aversion (CPA). The results showed that CCK2 receptor antagonist (LY-288,513, 10 µg, i.c.v.), but not CCK1 receptor antagonist (L-364,718, 10 µg, i.c.v.), inhibited the acquisition of CPA when given prior to naloxone (0.3 mg/kg) administration in morphine-dependent rats. Similarly, CCK-8 (0.1–1 µg, i.c.v.) significantly attenuated naloxone-precipitated withdrawal-induced CPA, and this inhibitory function was blocked by co-injection with L-364,718. Microinjection of L-364,718, LY-288,513 or CCK-8 to saline pretreated rats produced neither a conditioned preference nor aversion, and the induction of CPA by CCK-8 itself after morphine pretreatments was not significant. Our study identifies a different role of CCK1 and CCK2 receptors in negative affective components of morphine abstinence and an inhibitory effect of exogenous CCK-8 on naloxone-precipitated withdrawal-induced CPA via CCK1 receptor. PMID:22848639

  12. Lupus myocarditis: case report

    SciTech Connect

    LaManna, M.M.; Lumia, F.J.; Gordon, C.I.; Sumathisena; Maranhao, V.

    1988-03-01

    Although gallium-67 (/sup 67/Ga) accumulates in both neoplastic and inflammatory tissues, indium-111 (/sup 111/In) labeled leukocytes are seen only in inflammatory cells. Indium-111-labeled leukocytes therefore are a useful agent in the noninvasive differentiation of inflammatory tissue from neoplastic tissue. This case is an interesting example of the use of /sup 111/In-labeled leukocytes to make that differentiation.

  13. Diagnosis of osteomyelitis of the foot in diabetic patients: Value of 111In-leukocyte scintigraphy

    SciTech Connect

    Larcos, G.; Brown, M.L.; Sutton, R.T. )

    1991-09-01

    The noninvasive diagnosis of osteomyelitis of the foot in diabetic patients with currently available radiologic and radionuclide imaging techniques is often difficult. Recently, 111In-labeled leukocyte scintigraphy has been proposed as an attractive alternative. Accordingly, the authors retrospectively reviewed 51 111In-labeled leukocyte scans, 49 technetium-99m bone scans, and 49 plain radiographs obtained in 51 adults with diabetes in whom osteomyelitis of the foot was suspected. The sensitivity and specificity of these techniques were evaluated in all patients, as well as in a subgroup of 11 patients with neuroarthropathy. Results with 111In-labeled leukocyte scans were also examined in subsets of patients with soft-tissue ulcers (n = 35) and those receiving antibiotics during investigation (n = 20). Confirmation or exclusion of osteomyelitis was made surgically in 28 patients and clinically in 23. Fourteen patients had osteomyelitis. Bone scans were most sensitive (93%) but least specific (43%); plain radiographs were most specific (83%) but least sensitive (43%). 111In-labeled leukocyte scans were both sensitive (79%) and specific (78%), and remained useful in patients with neuroarthropathy, soft-tissue ulcers, and antibiotic treatment. Poor spatial resolution contributed to the false-negative and false-positive 111In-labeled leukocyte scans, suggesting that this technique should not be interpreted independent of other tests. 111In-labeled leukocyte scans are a valuable diagnostic tool for the diagnosis of pedal osteomyelitis in diabetic patients.

  14. Different peripheral and central antagonistic activity of new glutaramic acid derivatives on satiety induced by cholecystokinin in rats.

    PubMed

    Makovec, F; Bani, M; Chistè, R; Revel, L; Rovati, L C; Setnikar, I

    1986-12-30

    New glutaramic acid derivatives with cholecystokinin antagonistic activity were evaluated for their capacity to inhibit the satiety effect induced in the rat by intraperitoneal (i.p.) injection of cholecystokinin octapeptide (CCK-8). The most active compound, CR 1409, is about 4000 times more potent than proglumide when injected peripherally (i.p.). This compound competitively inhibits the action of CCK-8 at the receptor responsible for the satiety effect. In contrast, CR 1409, i.p. or intracerebroventricularly (i.c.v.) injected does not exhibit antagonistic effects when CCK-8 is administered i.c.v., confirming the existence of at least two different populations of CCK receptors.

  15. Cholecystokinin-octapeptide restored morphine-induced hippocampal long-term potentiation impairment in rats.

    PubMed

    Wen, Di; Zang, Guoqing; Sun, DongLei; Yu, Feng; Mei, Dong; Ma, Chunling; Cong, Bin

    2014-01-24

    Cholecystokinin-octapeptide (CCK-8), which is a typical brain-gut peptide, exerts a wide range of biological activities on the central nervous system. We have previously reported that CCK-8 significantly alleviated morphine-induced amnesia and reversed spine density decreases in the CA1 region of the hippocampus in morphine-treated animals. Here, we investigated the effects of CCK-8 on long-term potentiation (LTP) in the lateral perforant path (LPP)-granule cell synapse of rat dentate gyrus (DG) in acute saline or morphine-treated rats. Population spikes (PS), which were evoked by stimulation of the LPP, were recorded in the DG region. Acute morphine (30mg/kg, s.c.) treatment significantly attenuated hippocampal LTP and CCK-8 (1μg, i.c.v.) restored the amplitude of PS that was attenuated by morphine injection. Furthermore, microinjection of CCK-8 (0.1 and 1μg, i.c.v.) also significantly augmented hippocampal LTP in saline-treated (1ml/kg, s.c.) rats. Pre-treatment of the CCK2 receptor antagonist L-365,260 (10μg, i.c.v) reversed the effects of CCK-8, but the CCK1 receptor antagonist L-364,718 (10μg, i.c.v) did not. The present results demonstrate that CCK-8 attenuates the effect of morphine on hippocampal LTP through CCK2 receptors and suggest an ameliorative function of CCK-8 on morphine-induced memory impairment.

  16. CI-988 Inhibits EGFR Transactivation and Proliferation Caused by Addition of CCK/Gastrin to Lung Cancer Cells.

    PubMed

    Moody, Terry W; Nuche-Berenguer, Bernardo; Moreno, Paola; Jensen, Robert T

    2015-07-01

    Cholecystokinin (CCK) receptors are G-protein coupled receptors (GPCR) which are present on lung cancer cells. CCK-8 stimulates the proliferation of lung cancer cells, whereas the CCK2R receptor antagonist CI-988 inhibits proliferation. GPCR for some gastrointestinal hormones/neurotransmitters mediate lung cancer growth by causing epidermal growth factor receptor (EGFR) transactivation. Here, the role of CCK/gastrin and CI-988 on EGFR transactivation and lung cancer proliferation was investigated. Addition of CCK-8 or gastrin-17 (100 nM) to NCI-H727 human lung cancer cells increased EGFR Tyr(1068) phosphorylation after 2 min. The ability of CCK-8 to cause EGFR tyrosine phosphorylation was blocked by CI-988, gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), and tiron (superoxide scavenger). CCK-8 nonsulfated and gastrin-17 caused EGFR transactivation and bound with high affinity to NCI-H727 cells, suggesting that the CCK2R is present. CI-988 inhibited the ability of CCK-8 to cause ERK phosphorylation and elevate cytosolic Ca(2+). CI-988 or gefitinib inhibited the basal growth of NCI-H727 cells or that stimulated by CCK-8. The results indicate that CCK/gastrin may increase lung cancer proliferation in an EGFR-dependent manner.

  17. Reduction of food intake by central administration of cholecystokinin octapeptide in the rat is dependent upon inhibition of brain peptidases.

    PubMed Central

    Griesbacher, T.; Leighton, G. E.; Hill, R. G.; Hughes, J.

    1989-01-01

    1. The effects of intracerebroventricular (i.c.v.) injections of cholecystokinin-octapeptide (CCK-8) and caerulein, an amphibian decapeptide structurally related to CCK-8, are inconsistent in the rat. We have therefore investigated the possibility that enzymatic degradation could be responsible for the lack of activity of CCK-8 seen in some studies on food intake. 2. Injections of CCK-8 at doses of 2.5 nmol and 25 nmol into the lateral cerebral ventricle of rats did not reduce the intake of a highly palatable diet whereas injections of the same doses of caerulein reduced food intake potently and dose-dependently. 3. Co-administration of CCK-8 with a combination of the peptidase inhibitors bestatin (70 nmol), captopril (100 nmol) and thiorphan (120 nmol) resulted in an inhibition of feeding similar to that seen after the injection of caerulein alone. The peptidase inhibitors alone did not affect food intake. 4. When caerulein was injected i.c.v. in combination with bestatin, captopril and thiorphan the effect of caerulein was potentiated, suggesting that enzymatic breakdown of caerulein does occur. 5. It is concluded that the effect of centrally administered CCK-8 on food intake is dependent on the activity of cleaving enzymes in the brain. It is emphasized that the action of brain peptidases is a major factor which has to be considered when investigating the role of peptides in the central nervous system. PMID:2647203

  18. Murine eosinophils labeled with indium-111 oxine: localization to delayed hypersensitivity reactions against a schistosomal antigen and to lymphokine in vivo

    SciTech Connect

    Rand, T.H.; Clanton, J.A.; Runge, V.; English, D.; Colley, D.G.

    1983-04-01

    We have evaluated a method for quantitation of eosinophil migration to stimuli in vivo. Upon transfusion into normal syngeneic mice, 111In-labeled eosinophils had an intravascular half-life of 9.5 hr and distributed predominantly into spleen, bone marrow, and liver. In either Schistosoma mansoni-infected mice or recipients of lymphoid cells from infected mice, intradermal (ear pinna) injection of the schistosomal egg antigenic preparation (SEA) elicited time-dependent accumulation of 111In-labeled eosinophils detectable by either gamma scintillation counting of tissue samples or by nuclear medicine external imaging. Intradermal administration of a lymphokine fraction (containing eosinophil stimulation promoter activity) similarly caused accumulation of 111In-labeled eosinophils. Both reactions depended on the concentration of stimulus (SEA or lymphokine). 111In-labeled neutrophils or macrophages or 125I-albumin did not preferentially accumulate at the reactions examined to the extent found with 111In-labeled eosinophils, indicating that localization of label depends on an active process and is due to eosinophils rather than a contaminating cell type. The method was used to estimate how long eosinotactic lymphokine remained at dermal sites: 60% of initial activity was present 12 hr after injection. The model is discussed with regard to the role of lymphokines in hypersensitivity reactions with eosinophil involvement, such as the granulomatous response to S. mansoni eggs.

  19. Osteomyelitis complicating fracture: pitfalls of /sup 111/In leukocyte scintigraphy

    SciTech Connect

    Kim, E.E.; Pjura, G.A.; Lowry, P.A.; Gobuty, A.H.; Traina, J.F.

    1987-05-01

    /sup 111/In-labeled leukocyte imaging has shown greater accuracy and specificity than alternative noninvasive methods in the detection of uncomplicated osteomyelitis. Forty patients with suspected osteomyelitis complicating fractures (with and without surgical intervention) were evaluated with /sup 111/In-labeled leukocytes. All five patients with intense focal uptake, but only one of 13 with no uptake, had active osteomyelitis. However, mild to moderate /sup 111/In leukocyte uptake, observed in 22 cases, indicated the presence of osteomyelitis in only four of these; the other false-positive results were observed in noninfected callus formation, heterotopic bone formation, myositis ossificans, and sickle-cell disease. These results suggest that /sup 111/In-labeled leukocyte imaging is useful for the evaluation of suspected osteomyelitis complicating fracture but must be used in conjunction with clinical and radiographic correlation to avoid false-positive results.

  20. Visualization of a prosthetic vascular graft due to platelet contamination during /sup 111/Indium-labeled leukocyte scintigraphy

    SciTech Connect

    Oates, E.; Ramberg, K.

    1988-09-01

    A prosthetic axillo-femoral bypass graft was visualized during /sup 111/In-labeled leukocyte scintigraphy in a patient referred for possible abdominal abscess. The presence of significant cardiac blood-pool activity raised the possibility that this uptake was due to deposition of contaminating labeled platelets rather than labeled leukocytes. An analysis of a small sample of the patient's blood confirmed that the circulating activity was due to labeled platelets. Increased activity along prosthetic vascular grafts in patients undergoing /sup 111/In-labeled leukocyte scintigraphy may be due to adherent platelet, and not indicative of infection.

  1. Edwardsiella tarda Endocarditis Confirmed by Indium-111 White Blood Cell Scan: An Unusual Pathogen and Diagnostic Modality.

    PubMed

    Litton, Kayleigh M; Rogers, Bret A

    2016-01-01

    Edwardsiella tarda is a freshwater marine member of the family Enterobacteriaceae which often colonizes fish, lizards, snakes, and turtles but is an infrequent human pathogen. Indium-111- ((111)In-) labeled white blood cell (WBC) scintigraphy is an imaging modality which has a wide range of reported sensitivity and specificity (from 60 to 100% and from 68 to 92%, resp.) for diagnosing acute and chronic infection. We describe a case of suspected E. tarda prosthetic aortic valve and mitral valve endocarditis with probable vegetations and new mitral regurgitation on transthoracic and transesophageal echocardiograms which was supported with the use of (111)In-labeled WBC scintigraphy.

  2. Topical cholecystokinin depresses itch-associated scratching behavior in mice.

    PubMed

    Fukamachi, Shoko; Mori, Tomoko; Sakabe, Jun-Ichi; Shiraishi, Noriko; Kuroda, Etsushi; Kobayashi, Miwa; Bito, Toshinori; Kabashima, Kenji; Nakamura, Motonobu; Tokura, Yoshiki

    2011-04-01

    Cholecystokinin (CCK) serves as a gastrointestinal hormone and also functions as a neuropeptide in the central nervous system (CNS). CCK may be a downregulator in the CNS, as represented by its anti-opioid properties. The existence of CCK in the peripheral nervous system has also been reported. We investigated the suppressive effects of various CCKs on peripheral pruritus in mice. The clipped backs of ICR mice were painted with CCK synthetic peptides and injected intradermally with substance P (SP). The frequency of SP-induced scratching was reduced significantly by topical application of sulfated CCK8 (CCK8S) and CCK7 (CCK7S), but not by nonsulfated CCK8, CCK7, or CCK6. Dermal injection of CCK8S also suppressed the scratching frequency, suggesting that dermal cells as well as epidermal keratinocytes (KCs) are the targets of CCKs. As determined using real-time PCR, mRNA for CCK2R, one of the two types of CCK receptors, was expressed highly in mouse fetal skin-derived mast cells (FSMCs) and moderately in ICR mouse KCs. CCK8S decreased in vitro compound 48/80-promoted degranulation of FSMCs with a transient elevation of the intracellular calcium concentration. These findings suggest that CCK may exert an antipruritic effect via mast cells and that topical CCK may be clinically useful for pruritic skin disorders.

  3. Copper ions interfere with the reduction of the water-soluble tetrazolium salt-8.

    PubMed

    Semisch, Annetta; Hartwig, Andrea

    2014-02-17

    Metabolic activity as a measure of cell viability is frequently determined using the water-soluble tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), commercially available as CCK-8 reagent. In this study, CCK-8 was investigated with respect to its suitability for investigating nano- and microscale copper oxide (CuO NP and CuO MP) as well as water-soluble copper chloride (CuCl2). The results were compared to cell number and colony forming ability. Our data demonstrate that the CCK-8 assay overestimates the loss of metabolic activity by CuCl2 and CuO NP, because of interference by copper ions with the reduction of the dye.

  4. Positive indium-111 leukocyte scan in Nocardia brain abscess

    SciTech Connect

    Bauman, J.M.; Osenbach, R.; Hartshorne, M.F.; Youngblood, L.; Crooks, L.; Landry, A.J.; Cawthon, M.A.

    1986-01-01

    We report a case of clinically unsuspected nocardia brain abscess detected by /sup 111/In-labeled autologous leukocytes. Clinical and computed tomographic findings supported the diagnosis of primary or metastatic tumor and the patient was treated with dexamethasone for 30 days prior to the leukocyte scan. Labeled leukocytes may provide a sensitive discriminator for brain abscess despite previous therapy with steroids.

  5. Relationship between in vitro binding activity and in vivo tumor accumulation of radiolabeled monoclonal antibodies

    SciTech Connect

    Sakahara, H.; Endo, K.; Koizumi, M.; Nakashima, T.; Kunimatsu, M.; Watanabe, Y.; Kawamura, Y.; Nakamura, T.; Tanaka, H.; Kotoura, Y.

    1988-02-01

    The relationship between in vitro cell binding and in vivo tumor accumulation of radiolabeled antibodies was studied using /sup 125/I- and /sup 111/In-labeled monoclonal antibodies to human osteosarcoma, and a human osteosarcoma xenograft (KT005) in nude mice. Three monoclonal antibodies--OST6, OST7, and OST15--raised against human osteosarcoma recognize the same antigen molecule. Although the binding of both /sup 125/I- and /sup 111/In-labeled OST6 to KT005 cells was higher than that of radiolabeled OST7 in vitro, /sup 125/I-labeled OST6 showed a faster clearance from the circulation and a lower accumulation in the transplanted tumor than /sup 125/I-labeled OST7. In contrast to the radioiodinated antibodies, the in vivo tumor accumulation of /sup 111/In-labeled OST6 was higher, although not significantly, than that of /sup 111/In-labeled OST7. OST15 showed the lowest binding in vitro, and its in vivo tumor localization was also lower than the others. The discrepancy in tumor uptake between OST6 and OST7 labeled with either /sup 125/I or /sup 111/In may have been a result of differing blood clearance. These results suggest that binding studies can be used to exclude from in vivo use those antibodies which show very poor binding in vitro, while in vivo serum clearance may be a better test for choosing antibodies with similar binding.

  6. Involvement of cholecystokinin in peripheral nociceptive sensitization during diabetes in rats as revealed by the formalin response.

    PubMed

    Juárez-Rojop, Isela E; Granados-Soto, Vinicio; Díaz-Zagoya, Juan C; Flores-Murrieta, Francisco J; Torres-López, Jorge E

    2006-05-01

    The possible pronociceptive role of peripheral cholecystokinin (CCK-8) as well as CCK(A) and CCK(B) receptors in diabetic rats was assessed. Subcutaneous injection of 0.5% formalin induced a greater nociceptive behavior in diabetic than in non-diabetic rats. Moreover, local peripheral injection of CCK-8 (0.1-100 microg) significantly increased 0.5% formalin-induced nociceptive activity in diabetic, but not in non-diabetic, rats. This effect was restricted to the formalin-injected paw as administration of CCK-8 into the contralateral paw was ineffective. Local peripheral administration of CCK-8, in the absence of formalin injection, produced a low level of, but significant increase in, flinching behavior in diabetic compared to non-diabetic rats. In addition, local peripheral administration of the non-selective CCK receptor antagonist proglumide (1-100 microg), CCK(A) receptor antagonist lorglumide (0.1-100 microg) or CCK(B) receptor antagonist CR-2945 (0.1-100 microg), but not vehicle or contralateral administration of CCK receptor antagonists, significantly reduced 0.5% formalin-induced flinching in diabetic rats. CR-2945 was the most effective drug in this condition. These effects were not observed in non-diabetic rats. The local peripheral pronociceptive effect of CCK-8 (100 microg) was significantly reduced by proglumide (100 microg), lorglumide (100 microg), and CR-2945 (100 microg). Results suggest that diabetes-induced peripheral sensitization could be due to a local peripheral release of CCK-8, which in turn would act on CCK(B), mainly but also in CCK(A), receptors located on the primary afferent neurons.

  7. Activation of enteric nerve pathways in the guinea-pig duodenum by cholecystokinin octapeptide and pentagastrin.

    PubMed Central

    Ngu, M C

    1985-01-01

    The action and mechanism of action of cholecystokinin octapeptide (CCK-8) and pentagastrin on isolated segments of guinea-pig duodenum were examined using contractility studies and by intracellular recordings made from smooth muscle cells. Both CCK-8 and pentagastrin caused an excitatory contractile response. The threshold concentration ranged from 5 X 10(-11) to 10(-9) M for CCK-8 and 5 X 10(-10) to 10(-8) M for pentagastrin. The excitatory response was abolished by tetrodotoxin (3.1 X 10(-6) M) and atropine (1.5 X 10(-6) M) and inhibited by d-tubocurarine (up to 2.9 X 10(-5) M). In the presence of atropine a proportion of preparations relaxed in response to CCK-8 (nineteen of thirty-one) and pentagastrin (thirteen of seventeen). This response was only seen at high concentrations of the peptides (10(-8)-10(-7) M) and was abolished by tetrodotoxin (3 X 10(-6) M). Intracellular recordings from duodenal smooth muscle revealed multiple excitatory junction potentials (e.j.p.s) in response to CCK-8 and to pentagastrin. These e.j.p.s were identical to those evoked by transmural nerve stimulation and were abolished by atropine (1.5 X 10(-7) M) and by tetrodotoxin (3 X 10(-6) M). Inhibitory junction potentials (i.j.p.s) were not recorded in response to the peptides except on one occasion. It is suggested that CCK-8 and pentagastrin cause an increase in duodenal motility by the selective activation of excitatory pathways in the enteric nervous system. PMID:4032301

  8. Lack of interaction between cholecystokinin and opioid systems in the central control of breathing.

    PubMed

    Dierssen, M; Flórez, J; Hurlé, M A

    1989-01-01

    The present study investigates the interaction between CCK and opioid systems on the central control of breathing by analyzing the respiratory effects of CCK-8S (0.09 nmol) and met-enkephalin (0.7 and 1.4 mumol) applied to the ventral medullary surface of cats under the influence of the opiate antagonist naloxone and the CCK antagonist proglumide. Neither naloxone nor proglumide were able to modify the respiratory effects of CCK-8S and met-enkephalin, respectively. Therefore, the hypothesis that CCK and the opioid systems might operate as parallel and antagonic forces throughout the central nervous system, is no longer tenable.

  9. Differentiation of central and peripheral cholecystokinin receptors by new glutaramic acid derivatives with cholecystokinin-antagonistic activity.

    PubMed

    Makovec, F; Bani, M; Chisté, R; Revel, L; Rovati, L C; Rovati, L A

    1986-01-01

    Three glutaramic acid derivatives provided with a potent antagonistic activity on the contractions elicited by the carboxyl terminal octapeptide CCK-8 in the guinea pig gallbladder have been evaluated for their capacity to inhibit the binding of [125I]-(Bolton-Hunter)-CCK-8 to both central and peripheric cholecystokinin (CCK) receptors. The most active compound inhibits the CCK binding to rat pancreas acini at a concentration 10(-7) mol/l, but only at 10(-4) mol/l on cerebral cortex membranes, confirming the existence of at least two different populations of CCK receptors.

  10. Peptide-Conjugated Polymeric Micellar Nanoparticles for Dual SPECT and Optical Imaging of EphB4 Receptors in Prostate Cancer Xenografts

    PubMed Central

    Zhang, Rui; Xiong, Chiyi; Huang, Miao; Zhou, Min; Huang, Qian; Wen, Xiaoxia; Liang, Dong; Li, Chun

    2011-01-01

    EphB4, a member of the largest family of receptor tyrosine kinases, is overexpressed in numerous tumors. In this study, we developed a new class of multimodal nanoplatform for dual single photon emission computed tomography (SPECT) and near-infrared fluorescence imaging of EphB4. EphB4-binding peptide TNYL-FSPNGPIARAW (TNYL-RAW) was conjugated to polyethylene glycol-coated, core-crosslinked polymeric micelles (CCPM) dually labeled with near-infrared fluorescence fluorophores (Cy7) and a radioisotope (indium 111). In vitro, TNYL-RAW-CCPM selectively bound to EphB4-positive PC-3M prostate cancer cells, but not to EphB4-negative A549 lung cancer cells. In vivo, PC-3M tumors were clearly visualized by both SPECT and near-infrared fluorescence tomography after intravenous administration of 111In-labeled TNYL-RAW-CCPM. In contrast, there was little signal in A549 tumors of mice injected with 111In-labeled TNYL-RAW-CCPM or in PC-3M tumors of mice injected with 111In-labeled CCPM. The high accumulation of 111In-labeled TNYL-RAW-CCPM in PC-3M tumor could be significantly reduced after co-injection with an excess amount of TNYL-RAW peptide. Immunohistochemical analysis showed that fluorescence signal from the nanoparticles correlated with their radioactivity count, and co-localized with the EphB4 expressing region. 111In-labeled TNYL-RAW-CCPM allowed visualization of cancer cells overexpressing EphB4 by both nuclear and optical techniques. The complementary information acquired with multiple imaging techniques should be advantageous in early detection of cancer. PMID:21612822

  11. Effects of fasting and refeeding on the digestive tract of zebrafish (Danio rerio) fed with Spirulina (Arthrospira platensis), a high protein feed source.

    PubMed

    Lo Cascio, Patrizia; Calabrò, Concetta; Bertuccio, Clara; Paterniti, Irene; Palombieri, Deborah; Calò, Margherita; Albergamo, Ambrogina; Salvo, Andrea; Gabriella Denaro, Maria

    2017-07-01

    In the present work, morphological and molecular effects of short-term feed deprivation and refeeding with Spirulina (Arthrospira platensis) on zebrafish digestive tract were determined. Once elucidated the proximate composition of Spirulina feed, immunohistochemical and western blot analyses of peptide transporter (PepT1) and cholecystokinin (CCK8) were carried out in the gastrointestinal tract of zebrafish, previously morphologically investigated. Two and five fasting days caused not only morphostructural alterations, but also the downregulation of PepT1 and CCK8 proteins. Conversely, the recovery of normal morphological conditions, along with an increased PepT1 and CCK8 expression, were observed after refeeding with Spirulina. The increase of PepT1 expression in zebrafish may be responsible for the enhanced CCK8 secretion, so that both proteins may contribute to an improved digestion process during refeeding. These observations could be supported not only by compensatory mechanisms induced by fasting and refeeding but also by an higher protein quality of Spirulina-based diet.

  12. Distribution of neuroendocrine cells in the small and large intestines of the one-humped camel (Camelus dromedarius).

    PubMed

    Ali, M Al Haj; Nyberg, Fred; Chandranath, S I; Dhanasekaran, S; Tariq, Saeed; Petroianu, G; Hasan, M Y; Adeghate, Ernest A; Adem, A

    2007-10-01

    The distribution and relative frequency of neuroendocrine cells in the small and large intestines of one-humped camel were studied using antisera against 5-hydroxytryptamine (5-HT), cholecystokinin (CCK-8), somatostatin (SOM), peptide tyrosine tyrosine (PYY), gastric inhibitory polypeptide (GIP), neuronal nitric oxide synthase (nNOS), gastrin releasing peptide (GRP), substance P (SP), and neurokinin A (NKA). Among these cell types, CCK-8 immunoreactive (IR) cells were uniformly distributed in the mucosa, while others showed varied distribution in the villi or crypts of the small intestine. Immunoreactive cells like 5HT, CCK-8, and SOM showed peak density in the villi and crypts of the small intestine and in the colonic glands of the large intestine, while cells containing SP were discerned predominately in the crypts. 5-HT, CCK-8 and SOM cells were mainly flask-shaped and of the open-variety, while PYY and SP immunoreactive cells were mainly rounded or basket-shaped and of the closed variety. Basically the distribution pattern of the endocrine cells in the duodenum, jejunum and colon of the one-humped camel is similar to that of other mammals. Finally, the distribution of these bioactive agents may give clues as to how these agents aid in the function of the intestinal tract of this desert animal.

  13. Peripheral participation of cholecystokinin in the morphine-induced peripheral antinociceptive effect in non-diabetic and diabetic rats.

    PubMed

    Torres-López, Jorge E; Juárez-Rojop, Isela E; Granados-Soto, Vinicio; Diaz-Zagoya, Juan C; Flores-Murrieta, Francisco J; Ortíz-López, José U S; Cruz-Vera, Jorge

    2007-03-01

    The effects of cholecystokinin (CCK-8) and the CCK receptor antagonist proglumide, on antinociception induced by local peripheral (subcutaneous) injected morphine in non-diabetic (ND) and streptozotocin-induced diabetic (D) rats, were examined by means of the formalin test. Morphine induced dose-dependent antinociception both in ND and D rats. However, in D rats, antinociceptive morphine potency was about twofold less than in ND rats. Pre-treatment with CCK-8 abolished the antinociceptive effect of morphine in a dose-dependent manner in both groups of rats. Additionally, proglumide enhanced the antinociceptive effect induced by all doses of morphine tested. Both CCK-8 and proglumide had no effect on flinching behaviour when given alone to ND rats. Unlike ND rats, in D rats proglumide produced dose-dependent antinociception and CCK-8 enhanced formalin-evoked flinches, as observed during the second phase of the test. In conclusion, our data show a decrease in peripheral antinociceptive potency of morphine when diabetes was present. Additionally, peripheral CCK plays an antagonic role to the peripheral antinociceptive effect of morphine, additional to the well known CCK/morphine interaction at spinal and supraspinal level.

  14. Characterization of a new CCK antagonist, L364,718: In vitro and in vivo studies

    SciTech Connect

    Louie, D.S.; Liang, Jiang Ping; Owyang, Chung )

    1988-09-01

    In this study the authors examined a novel, orally effective, nonpeptidal cholecystokinin (CCK) antagonist, 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-1H-indole-2-carboxamide (L364,718) on CCK-induced amylase release. They used isolated rat pancreatic acini and incubated them with CCK-8 with or without various CCK receptor antagonists. L364,718, proglumide, and the proglumide derivative CR1409 each caused a progressive rightward shift in the CCK-8-dose-response curve without a change in maximal amylase secretion. L364,718 was 600-fold more potent than CR1409 and 2,000,000-fold more potent than proglumide in inhibiting CCK-8-induced amylase release. Inhibition of {sup 125}I-Bolton-Hunter-CCK-8 binding to acini by these receptor antagonists had a similar rank potency. L364,718 was tested against other pancreatic exocrine secretagogues and was effective against agonists that only act through the CCK receptor. To verify that L364,718 is an effective receptor antagonists against the various molecular forms of CCK released endogenously in humans, postprandial plasma CCK was extracted and bioassayed using amylase release from isolated pancreatic acini. Thus L364,718 is the most potent, selective peripheral CCK receptor antagonist reported to data, and it is capable of antagonizing the stimulatory action of exogenously as well as endogenously released CCK to evoke amylase release from pancreatic acini.

  15. The effect of cholecystokinin on intracellular Ca2+, membrane-associated protein kinase-C activity, and progesterone production in chicken granulosa cells.

    PubMed

    Morley, P; Wang, J; Vanderhyden, B C; Chakravarthy, B; Durkin, J; Whitefield, J F

    1993-11-01

    Nerve fibers immunoreactive for cholecystokinin (CCK) have been observed in the rat ovary, but the function of this gut peptide in the ovary is not known. These studies were designed to investigate the effects of the CCK C-terminal octapeptide (CCK-8) on the intracellular calcium ion concentration ([Ca2+]i), protein kinase-C (PKC) activity, and progesterone secretion in granulosa cells obtained from the two largest preovulatory follicles (F1 and F2) of hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 96 +/- 5 nM. There was a rapid (i.e. within 5-10 sec) 2- to 4-fold increase in [Ca2+]i in 70% of the cells examined after the addition of 10(-7) M CCK-8. The CCK-8-triggered [Ca2+]i transient was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating the cells with a Ca2+ channel blocker, such as La3+ (1 mM) or D600 (100 microM). The CCK-8-triggered [Ca2+]i surge was abolished by pretreating the cells with the inhibitor of inositol phospholipid hydrolysis, neomycin (1.5 mM), the CCK antagonists proglumide (1 mM) and benzotript (1 mM), or pertussis toxin (50 ng/ml for 12 h). Incubating granulosa cells with CCK-8 (2 x 10(-7) M) for 10 min stimulated a 1.60 +/- 0.04-fold increase in membrane-associated PKC activity over control levels. In 3-h incubations, CCK-8 (10(-6)-10(-8) M) did not affect basal or LH (20 or 100 ng/ml-stimulated progesterone production. These studies demonstrate that CCK-8 causes a transient increase in chicken granulosa cell [Ca2+]i through the release of Ca2+ from intracellular stores and activates membrane-associated PKC activity, but does not affect progesterone production. These results suggest the presence of G-protein-coupled phospholipase-C-activating CCK receptors on the surface of these cells.

  16. Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors.

    PubMed

    Smeets, R L; Fouraux, M A; van Emst-de Vries, S E; De Pont, J J; Willems, P H

    1998-03-01

    1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition

  17. Effect of peripheral administration of cholecystokinin on food intake in apolipoprotein AIV knockout mice

    PubMed Central

    Yoshimichi, Go; Lo, Chunmin C.; Tamashiro, Kellie L. K.; Ma, Liyun; Lee, Dana M.; Begg, Denovan P.; Liu, Min; Sakai, Randall R.; Woods, Stephen C.; Yoshimatsu, Hironobu

    2012-01-01

    Apolipoprotein AIV (apo AIV) and cholecystokinin (CCK) are satiation factors secreted by the small intestine in response to lipid meals. Apo AIV and CCK-8 has an additive effect to suppress food intake relative to apo AIV or CCK-8 alone. In this study, we determined whether CCK-8 (1, 3, or 5 μg/kg ip) reduces food intake in fasted apo AIV knockout (KO) mice as effectively as in fasted wild-type (WT) mice. Food intake was monitored by the DietMax food system. Apo AIV KO mice had significantly reduced 30-min food intake following all doses of CCK-8, whereas WT mice had reduced food intake only at doses of 3 μg/kg and above. Post hoc analysis revealed that the reduction of 10-min and 30-min food intake elicited by each dose of CCK-8 was significantly larger in the apo AIV KO mice than in the WT mice. Peripheral CCK 1 receptor (CCK1R) gene expression (mRNA) in the duodenum and gallbladder of the fasted apo AIV KO mice was comparable to that in WT mice. In contrast, CCK1R mRNA in nodose ganglia of the apo AIV KO mice was upregulated relative to WT animals. Similarly, upregulated CCK1R gene expression was found in the brain stem of apo AIV KO mice by in situ hybridization. Although it is possible that the increased satiating potency of CCK in apo AIV KO mice is mediated by upregulation of CCK 1R in the nodose ganglia and nucleus tractus solitarius, additional experiments are required to confirm such a mechanism. PMID:22461023

  18. Modulation of cytosolic free calcium levels by extracellular phosphate and lanthanum

    SciTech Connect

    Korc, M.; Schoeni, M.H.

    1987-03-01

    The effects of extracellular phosphate and lanthanum on cytosolic free Ca/sup 2 +/ ((Ca/sup 2 +/)/sub i/) levels were studied in isolated rat pancreatic acini. In the presence of 1.28 mM Ca/sup 2 +/ and 1.0 mM phosphate, the mean resting (Ca/sup 2 +/)/sub i/ level was 120 nM. Omission of phosphate from incubation medium significantly lowered this value to 94 nM. The gastrointestinal hormone cholecystokinin octapeptide (CCK-8) rapidly enhanced both (Ca/sup 2 +/)/sub i/ levels and /sup 45/Ca/sup 2 +/ efflux, irrespective of the presence or absence of phosphate. Lanthanum (0.1 mM), a compound known to block transmembrane Ca/sup 2 +/ fluxes, attenuated both actions of CCK-8, but only in the absence of extracellular phosphate. There was a concomitant decrease in amylase secretion induced by 0.1 nM CCK-8 but not by 10 nM CCK-8, without a significant change in cellular ATP levels. The inhibitory actions of lanthanum on CCK-8-stimulated (Ca/sup 2 +/)/sub i/ levels were very rapid and were mimicked only by prolonged incubation of acini in Ca/sup 2 +/-free medium supplemented with EGTA. Omission of phosphate from incubation medium also lowered basal (Ca/sup 2 +/)/sub i/ levels in IM-9 lymphocytes. These findings suggest that extracellular phosphate may modulate resting (Ca/sup 2 +/)/sub i/ levels in pancreatic acini and other cell types and that mobilization of intracellular Ca/sup 2 +/ may partly depend on the availability of a lanthanum-sensitive pool of cell-surface Ca/sup 2 +/ that is not readily removed by EGTA.

  19. Intracellular mediators of Na -K pump activity in guinea pig pancreatic acinar cells

    SciTech Connect

    Hootman, S.R.; Ochs, D.L.; Williams, J.A.

    1985-10-01

    The involvement of CaS and cyclic nucleotides in neurohormonal regulation of Na -K -ATPase (Na -K pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by (3H)ouabain binding and by ouabain-sensitive YWRb uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of (TH)ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in CaS -free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular CaS , while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free CaS levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent CaS chelator. Basal intracellular CaS concentration ((CaS )i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively.

  20. Effect of peripheral administration of cholecystokinin on food intake in apolipoprotein AIV knockout mice.

    PubMed

    Yoshimichi, Go; Lo, Chunmin C; Tamashiro, Kellie L K; Ma, Liyun; Lee, Dana M; Begg, Denovan P; Liu, Min; Sakai, Randall R; Woods, Stephen C; Yoshimatsu, Hironobu; Tso, Patrick

    2012-06-01

    Apolipoprotein AIV (apo AIV) and cholecystokinin (CCK) are satiation factors secreted by the small intestine in response to lipid meals. Apo AIV and CCK-8 has an additive effect to suppress food intake relative to apo AIV or CCK-8 alone. In this study, we determined whether CCK-8 (1, 3, or 5 μg/kg ip) reduces food intake in fasted apo AIV knockout (KO) mice as effectively as in fasted wild-type (WT) mice. Food intake was monitored by the DietMax food system. Apo AIV KO mice had significantly reduced 30-min food intake following all doses of CCK-8, whereas WT mice had reduced food intake only at doses of 3 μg/kg and above. Post hoc analysis revealed that the reduction of 10-min and 30-min food intake elicited by each dose of CCK-8 was significantly larger in the apo AIV KO mice than in the WT mice. Peripheral CCK 1 receptor (CCK1R) gene expression (mRNA) in the duodenum and gallbladder of the fasted apo AIV KO mice was comparable to that in WT mice. In contrast, CCK1R mRNA in nodose ganglia of the apo AIV KO mice was upregulated relative to WT animals. Similarly, upregulated CCK1R gene expression was found in the brain stem of apo AIV KO mice by in situ hybridization. Although it is possible that the increased satiating potency of CCK in apo AIV KO mice is mediated by upregulation of CCK 1R in the nodose ganglia and nucleus tractus solitarius, additional experiments are required to confirm such a mechanism.

  1. Cholecystokinin but not ghrelin stimulates mucosal bicarbonate secretion in rat duodenum: independence of feeding status and cholinergic stimuli.

    PubMed

    Sjöblom, Markus; Lindqvist, Ramin; Bengtsson, Magnus W; Jedstedt, Gunilla; Flemström, Gunnar

    2013-05-10

    Cholecystokinin (CCK) is an important regulator of food digestion but its influence on small intestinal secretion has received little attention. We characterized effects of CCK-8, ghrelin and some related peptides on duodenal HCO3(-) secretion in vivo and demonstrated CCK-induced calcium signaling in acutely isolated enterocytes. A segment of proximal duodenum with intact blood supply was cannulated in situ in anaesthetized rats. Mucosal HCO3(-) secretion was continuously recorded (pH-stat). Peptides were administrated to the duodenum by close intra-arterial infusion. Clusters of duodenal enterocytes were attached to the bottom of a perfusion chamber. The intracellular calcium concentration ([Ca(2+)]i) was examined by dual-wavelength imaging. CCK-8 (3.0, 15 and 60 pmol/kg,h) caused dose-dependent increases (p<0.01) in duodenal alkaline secretion in both overnight fasted and continuously fed animals. The CCK1R-antagonist devazepide but neither the CCK2R-antagonist YMM022 nor the melatonin MT2-selective antagonist luzindole inhibited the rise in secretion. Atropine decreased sensitivity to CCK-8. The appetite-related peptide ghrelin was without effect on the duodenal secretion in fasted as well as fed animals. Superfusion with CCK-8 (1.0-50 nM) induced [Ca(2+)]i signaling in acutely isolated duodenal enterocytes. After an initial peak response, [Ca(2+)]i returned to near basal values within 3-5min. Devazepide but not YMM022 inhibited this [Ca(2+)]i response. Low doses of CCK-8 stimulate duodenal alkaline secretion and induce enterocyte [Ca(2+)]i signaling by an action at CCK1 receptors. The results point to importance of CCK in the rapid postprandial rise in mucosa-protective duodenal secretion.

  2. Biomedical research with cyclotron produced radionuclides. Progress report, February 1, 1981-December 31, 1981

    SciTech Connect

    Laughlin, J.S.

    1981-09-01

    Progress is reported in the following areas: evaluation of chemotherapeutic regimens in solid tumors using /sup 13/N-labelled amino acids; organ imaging with /sup 13/N-labelled L-amino acids; imaging with /sup 111/In-labelled-autologous platelets; synthesis and biological studies of /sup 111/In-labelled ammonia and L-amino acids; synthesis and evaluation for pancreatic imaging of /sup 11/C-labelled amino acides; radioisotope monitoring of myocardiol function; synthesis of /sup 11/C-labelled precursor compounds; reduction of radiation exposure through automation and remote control; development of an anhydrous /sup 18/F target; evaluation of radiolabelled 5-fluorouracils for scintigraphy; and methods of data analysis, modeling, and unproving instrumentation for positron-emission tomography. (EDB)

  3. Indium-111-labeled leukocyte scintigraphy in hemodialysis access-site infection

    SciTech Connect

    Palestro, C.J.; Vega, A.; Kim, C.K.; Vallabhajosula, S.; Goldsmith, S.J. )

    1990-03-01

    Bacterial sepsis, a significant complication of chronic hemodialysis, is generally the result of infection at the vascular access site. We retrospectively reviewed the utility of indium-111-(111In) labeled autologous leukocyte scintigraphy in 26 patients (30 scans) with synthetic vascular grafts, on chronic hemodialysis, in whom hemodialysis access site infection was a diagnostic consideration. Leukocyte scintigraphy correctly identified all fifteen access-site infections; there was one false-positive study, for an overall sensitivity and specificity of 100% and 93%, respectively. Of particular significance is the fact that in nine (60%) of the fifteen access-site infections, physical examination was normal. Our data indicate that 111In-labeled leukocyte scintigraphy is a useful procedure for the diagnosis of hemodialysis access-site infection, and it is especially valuable when physical examination of the access site is normal.

  4. Pharmacokinetics of internally labeled monoclonal antibodies as a gold standard: comparison of biodistribution of /sup 75/Se-, /sup 111/In-, and /sup 125/I-labeled monoclonal antibodies in osteogenic sarcoma xenografts in nude mice

    SciTech Connect

    Koizumi, M.; Endo, K.; Watanabe, Y.; Saga, T.; Sakahara, H.; Konishi, J.; Yamamuro, T.; Toyama, S.

    1989-04-01

    In order to know the true biodistribution of anti-tumor monoclonal antibodies, three monoclonal antibodies (OST6, OST7, and OST15) against human osteosarcoma and control antibody were internally labeled with 75Se by incubating (75Se)methionine and hybridoma cells. 75Se-labeled monoclonal antibodies were evaluated both in vitro and in vivo using the human osteogenic sarcoma cell line KT005, and the results were compared with those of 125I- and 111In-labeled antibodies. 75Se-, 125I- and 111In-labeled monoclonal antibodies had identical binding activities to KT005 cells, and the immunoreactivity was in the decreasing order of OST6, OST7, and OST15. On the contrary, in vivo tumor uptake (% injected dose/g) of 75Se- and 125I-labeled antibodies assessed using nude mice bearing human osteosarcoma KT005 was in the order of OST7, OST6, and OST15. In the case of 111In, the order was OST6, OST7, and OST15. High liver uptake was similarly seen with 75Se- and 111In-labeled antibodies, whereas 125I-labeled antibodies showed the lowest tumor and liver uptake. These data indicate that tumor targeting of antibody conjugates are not always predictable from cell binding studies due to the difference of blood clearance of labeled antibodies. Furthermore, biodistribution of both 111In- and 125I-labeled antibodies are not identical with internally labeled antibody. Admitting that internally labeled antibody is a ''gold standard'' of biodistribution of monoclonal antibody, high liver uptake of 111In-radiolabeled antibodies may be inherent to antibodies. Little, if any, increase in tumor-to-normal tissue ratios of antibody conjugates will be expected compared to those of 111In-labeled antibodies if stably coupled conjugates are administered i.v.

  5. Rapid and simple methods for labeling white blood cells and platelets with indium-111-oxine

    SciTech Connect

    Steffel, F.G.; Rao, S.A.

    1987-06-01

    Simple procedures in a kit form for labeling white blood cells (WBCs) and platelets with commercially available indium-111 (/sup 111/In)-oxine have been developed for the convenience of small community hospitals. The time required for the labeling procedure is less than 2 hr. The resulting scintigrams from the clinical studies in both WBCs and platelets showed that the /sup 111/In-labeled cells have a high degree of viability.

  6. Targeting of indium 111-labeled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: Imaging of tumors hosted in nude mice

    SciTech Connect

    Le Doussal, J.M.; Gruaz-Guyon, A.; Martin, M.; Gautherot, E.; Delaage, M.; Barbet, J. )

    1990-06-01

    Antibody conjugates were prepared by coupling F(ab')2 or Fab' fragments of an antibody specific for the human high molecular weight-melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the antibody conjugate mediated binding of the 111In-labeled haptens to melanoma cells. In vivo, it allowed specific localization of the haptens in A375 tumors. The bivalent hapten exhibited much higher efficiency at targeting 111In onto cells, both in vitro and in vivo. Antibody conjugate and hapten doses (2 micrograms and 1 pmol, respectively) and the delay between antibody conjugate and tracer injections (24 h) were adjusted to maximize tumor uptake (4% injected dose/g) and tumor to normal tissue contrast (greater than 3) obtained 3 h after injection of the 111In-labeled bivalent hapten. This two-step technique, when compared to direct targeting of 111In-labeled F(ab')2 fragments, provided lower localization of injected activity into the tumor (x 0.25), but higher tumor/tissue ratios, especially with respect to liver (x 7), spleen (x 8), and kidneys (x 10). In addition, high contrast images were obtained within 3 hours, instead of days. Thus, antibody conjugate-mediated targeting of small bivalent haptens, labeled with short half-life isotopes, is proposed as a general method for improving tumor radioimmunolocalization.

  7. Indium 111-labeled platelet kinetic studies and platelet-associated IgG in hairy cell leukemia

    SciTech Connect

    Panzer, S.; Lechner, K.; Neumann, E.; Meryn, S.; Haubenstock, A.

    1986-07-15

    In order to study the pathogenesis of thrombocytopenia in patients with hairy cell leukemia (HCL), levels of platelet-associated IgG (PAIgG), platelet life span (MLS), and the sequestration site of autologous /sup 111/In-labeled platelets were measured in nine patients with HCL. Splenectomized patients (n = 4) had a higher platelet count (x = 122.5 X 10(9)/l; range, 80-190 X 10(9)/l) as well as higher levels of PAIgG (x = 10.7%; range, 5.8-16.9%), than nonsplenectomized patients (platelets x = 76 X 10(9)/l, range 40-100 X 10(9)/l; PAIgG x = 3.2%, range 2.2-4.2%). A normal recovery of /sup 111/In-labeled platelets was found in splenectomized patients, whereas a very low recovery was observed in the nonsplenectomized group (x = 70.2%, range, 50-82.5%, versus x = 22.4%, range, 15-28.2%). The MLS was borderline normal in all patients. The site of sequestration was the spleen in nonsplenectomized patients. The low recovery of /sup 111/In-labeled platelets in nonsplenectomized patients suggests hypersplenism with pooling as a major cause of thrombocytopenia, in addition to impaired thrombocytopoiesis and possible immune-mediated platelet destruction.

  8. Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCKA receptor

    PubMed Central

    Smeets, R L L; Fouraux, M A; Pouwels, W; van Emst-de Vries, S E; Ronken, E; De Pont, J J H H M; Willems, P H G M

    1998-01-01

    Many G protein-coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCKA) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild-type (CCKAWT) and mutant (CCKAMT) receptor were stably expressed in Chinese hamster ovary (CHO) cells.Binding of [3H]-cholecystokinin-(26-33)-peptide amide (CCK-8) to membranes prepared from CHO-CCKAWT cells and CHO-CCKAMT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK-8, respectively).The dose-response curves for CCK-8-induced cyclic AMP accumulation and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO-CCKAMT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO-CCKAWT cells. This demonstrates that attenuation of CCK-8-induced activation of adenylyl cyclase and phospholipase C-β involves a staurosporine-sensitive kinase, which acts directly at the potential sites of PKC action on the CCKA receptor in CCK-8-stimulated CHO-CCKAWT cells.The potent PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), evoked a rightward shift of the dose-response curve for CCK-8-induced cyclic AMP accumulation in CHO-CCKAWT cells but not CHO-CCKAMT cells. This is in agreement with the idea that PKC acts directly at the CCKA receptor to attenuate adenylyl cyclase activation.In contrast, TPA evoked a rightward shift of the dose-response curve for CCK-8-induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high-level PKC activation inhibits CCK-8-induced Ins(1,4,5)P3 formation also at a post-receptor site.TPA inhibition of agonist-induced Ca2+ mobilization was only partly reversed in CHO-CCKAMT cells. TPA also inhibited Ca2+ mobilization in response to the G

  9. Multiple sources of 1,2-diacylglycerol in isolated rat pancreatic acini stimulated by cholecystokinin. Involvement of phosphatidylinositol bisphosphate and phosphatidylcholine hydrolysis.

    PubMed

    Matozaki, T; Williams, J A

    1989-09-05

    Changes in the cellular content of 1,2-diacylglycerol (DAG) in isolated rat pancreatic acini in response to agonist stimulation were studied using a sensitive mass assay. When acini were stimulated by 10 nM COOH-terminal cholecystokinin-octapeptide (CCK8), the increase in DAG was biphasic, consisting of an early peak at 5 s and a second, larger, gradual increase that was maximal by 15 min. The basal level of DAG in acini was 1.04 nmol/mg of protein, which was increased to 1.24 nmol/mg of protein at 5 s and 2.76 nmol/mg of protein at 30 min. In comparison, the increase in DAG stimulated by 30 pM CCK8, a submaximal concentration for amylase release, was monophasic, increasing without an early peak but sustained to 60 min. Other Ca2+-mobilizing secretagogues such as carbamylcholine and bombesin increased DAG in acini, whereas vasoactive intestinal peptide, which acts to increase cAMP, had no effect. Phorbol ester and Ca2+ ionophore also stimulated DAG production. Analysis of the mass level of inositol 1,4,5-trisphosphate (1,4,5-IP3) showed that the generation of 1,4,5-IP3 stimulated by 10 nM CCK8 peaked at 5 s, a finding consistent with the early peak of DAG. The basal level was 4.7 pmol/mg of protein, which was increased to 144.6 pmol/mg of protein at 5 s by 10 nM CCK8. The levels of 1,4,5-IP3 then returned toward basal in contrast to the gradual and sustained increase of DAG. The dose dependencies of 1,4,5-IP3 and DAG formation at 5 s with respect to CCK8 were almost identical. This suggests that phosphatidylinositol 4,5-bisphosphate hydrolysis is a major source of the early increase in DAG but not of the sustained increase in DAG. Therefore, a possible contribution of phosphatidylcholine hydrolysis to DAG formation was examined utilizing acini prelabeled with [3H]choline. CCK8 (1 nM) maximally increased [3H]choline metabolite release by 133% of control at 30 min. Separation of these metabolites by thin layer chromatography showed that the products of CCK8

  10. In vitro transport and satiety of a beta-lactoglobulin dipeptide and beta-casomorphin-7 and its metabolites.

    PubMed

    Osborne, Simone; Chen, Wei; Addepalli, Rama; Colgrave, Michelle; Singh, Tanoj; Tran, Cuong; Day, Li

    2014-11-01

    Understanding the digestive behaviour and biological activities of dairy proteins may help to develop model dairy products with targeted health outcomes including increased satiety and healthy weight maintenance. Caseins and whey proteins constitute over 95% of milk proteins with consumption of these proteins associated with increased satiety and a decreased prevalence of metabolic disorders. To investigate the in vitro digestive behaviour and satiety of dairy proteins at the intestinal epithelium, the in vitro transport and hydrolysis of 500-2000 μM β-casomorphin-7 (YPFPGPI or β-CM7) and a β-lactoglobulin (β-Lg) dipeptide (YL) was measured using Caco-2 cell monolayers grown on transwells as a model of the intestinal epithelium. Transport of YL was concentration dependent and ranged from 0.37-5.26 × 10(-6) cm s(-1), whereas transport of β-CM7 was only detected at 2000 μM and was significantly lower at 0.13 × 10(-6) cm s(-1). Rapid hydrolysis of β-CM7 in the apical chamber by the Caco-2 cells produced three peptide metabolites: YP, GPI and FPGPI. All of these metabolites were detected in the basolateral chamber after 30 min with both the YP and GPI peptides transporting at a higher rate than intact β-CM7. In vitro satiety was indicated by the secretion of cholecystokinin [26-33] (CCK-8) and glucagon-like peptide 1 (GLP-17-36NH2) in the STC-1 enteroendocrine cell model. CCK-8 secretion was highest in response to β-CM7 followed by the β-CM7 metabolite FPGPI. CCK-8 secretion however was not significantly stimulated by the tri- or dipeptides. Secretion of GLP-1 was not significantly stimulated by β-CM7 or YL. These in vitro results suggest that dairy peptide size enhances CCK-8 secretion, whilst limiting transport across Caco-2 monolayers.

  11. Effect of cholecystokinin on experimental neuronal aging

    PubMed Central

    Sun, Xiao-Jiang; Lu, Qin-Chi; Cai, Yan

    2005-01-01

    AIM: To observe the effect of cholecystokinin (CCK) on lipofusin value, neuronal dendrite and spine ultrastructure, and total cellular protein during the process of experimental neuronal aging. METHODS: Experimental neuronal aging study model was established by NBA2 cellular serum-free culture method. By using single intracellular lipofusin value from microspectrophotometry, morphology of neuronal dendrites and spines from the scanner electron microscopy, and total cellular protein as the indexes of experimental neuronal aging, we observed the effect of CCK8 on the process of experimental neuronal aging. RESULTS: Under the condition of serum-free culture, intracellular fluorescence value (%) increased with the extension of culture time (1 d 8.51±3.43; 5 d 10.12±3.03; 10 d 20.54±10.3; 15 d 36.88±10.49; bP<0.01). When CCK was added to serum-free culture medium, intracellular lipofusin value (%) decreased remarkably after consecutive CCK reaction for 10 and 15 d (control 36.88±10.49; 5 d 32.03±10.01; 10 d 14.37±5.55; 15 d 17.31±4.80; bP<0.01). As the time of serum-free culturing was prolonged, the number of neuronal dendrite and spine cells decreased. The later increased in number when CCK8 was added. CCK8 could improve the total cellular protein in the process of experimental neuronal aging. CONCLUSION: CCK8 may prolong the process of experimental neuronal aging by maintaining the structure and the number of neuronal dendrite and spine cells and changing the total cellular protein. PMID:15641144

  12. Peripheral obestatin has no effect on feeding behavior and brain Fos expression in rodents

    PubMed Central

    Kobelt, Peter; Wisser, Anna-Sophia; Stengel, Andreas; Goebel, Miriam; Bannert, Norbert; Gourcerol, Guillaume; Inhoff, Tobias; Noetzel, Steffen; Wiedenmann, Bertram; Klapp, Burghard F.; Taché, Yvette; Mönnikes, Hubert

    2009-01-01

    Obestatin is produced in the stomach from proghrelin by post-translational cleavage. The initial report claimed anorexigenic effects of obestatin in mice. Contrasting studies indicated no effect of obestatin on food intake (FI). We investigated influences of metabolic state (fed/fasted), environmental factors (dark/light phase) and brain Fos response to intraperitoneal (ip) obestatin in rats, and used the protocol from the original study assessing obestatin effects in mice. FI was determined in male rats injected ip before onset of dark or light phase, with obestatin (1 or 5 μmol/kg), CCK8S (3.5 nmol/kg) or 0.15 M NaCl, after fasting (16 h, n = 8/group) or ad libitum (n = 10-14/group) food intake. Fos expression in hypothalamic and brainstem nuclei was examined in freely fed rats 90 min after obestatin (5 μmol/kg), CCK8S (1.75 nmol/kg) or 0.15 M NaCl (n = 4/group). Additionally, fasted mice were injected ip with obestatin (1 μmol/kg) or urocortin 1 (2 nmol/kg) 15 min before food presentation. No effect on FI was observed after obestatin administration during the light and dark phase under both metabolic conditions while CCK8S reduced FI irrespectively of the conditions. The number of Fos positive neurons was not modified by obestatin while CCK8S increased Fos expression in selective brain nuclei. Obestatin did not influence the refeeding response to a fast in mice, while urocortin was effective. Therefore, peripheral obestatin has no effect on FI under various experimental conditions and did not induce Fos in relevant central neuronal circuitries modulating feeding in rodents. PMID:18342400

  13. Dual effects of chlorobutanol on secretory response and intracellular Ca2+ dynamics in isolated pancreatic acini of the rat.

    PubMed Central

    Habara, Y.; Kanno, T.

    1993-01-01

    1. The effects of chlorobutanol, a widely used drug preservative, on exocrine response and intracellular Ca2+ dynamics were examined in isolated pancreatic acini of the rat. 2. Chlorobutanol (1 mg ml-1) markedly inhibited the secretory response to cholecystokinin octapeptide (CCK-8), carbamylcholine chloride (carbachol), or sodium fluoride, a direct G-protein activator. However, chlorobutanol itself induced a maximal release of amylase when the dose was increased to 4 mg ml-1. 3. An oscillatory fluctuation of cytoplasmic Ca2+ concentration, [Ca2+]c, induced by 5 pM CCK-8 or 0.3 microM carbachol was totally abolished in the presence of 1 mg ml-1 chlorobutanol. 4. A biphasic change in [Ca2+]c induced by 100 pM CCK-8, a rapid rise followed by a gradual decay, was transformed to an oscillatory fluctuation by the preservative. 5. Chlorobutanol inhibited 13 pM [125I]-CCK-8 or 0.5 nM [3H]-methylscopolamine chloride binding to the acinar cells in a dose-dependent manner. 6. These results indicate that chlorobutanol produces discernible pharmacological effects on the secretory response in rat pancreatic acinar cells through changes in the Ca2+ dynamics. Possible sites of action could be at a binding process of secretagogues to their receptors, at an activation process of a G-protein located in the plasma membrane, or at the processes following G-protein activation. However, the possibility that the preservative may distort the Ca(2+)-transport function of the plasma membrane or the membrane of intracellular organella, especially Ca(2+)-sequestering pools, cannot be excluded. PMID:7689400

  14. A new androstanoid metabolite from a soil fungus Curvularia borreriae strain HS-FG-237.

    PubMed

    Liu, Li-Fei; Zhang, Hui; Qi, Huan; Wang, Xiao-Ming; Wang, Ji-Dong; Tan, Gui-Shan

    2017-05-01

    A new androstanoid metabolite, 4α-methyl-9α-methoxyandrosta-8,15-diene-3,17-dione (1), was isolated from a soil fungus Curvularia borreriae (Pleosporaceae) strain HS-FG-237. Its structure was determined by extensive spectroscopic and X-ray diffraction analyses. Compound 1 exhibited poor cytotoxicity toward HCT-116 cells by CCK-8 assay and weak anti-inflammatory activity in an ANA-1 murine macrophages model.

  15. Role of capsaicin-sensitive peripheral sensory neurons in anorexic responses to intravenous infusions of cholecystokinin, peptide YY-(3-36), and glucagon-like peptide-1 in rats.

    PubMed

    Reidelberger, Roger; Haver, Alvin; Anders, Krista; Apenteng, Bettye

    2014-10-15

    Cholecystokinin (CCK)-induced suppression of feeding is mediated by vagal sensory neurons that are destroyed by the neurotoxin capsaicin (CAP). Here we determined whether CAP-sensitive neurons mediate anorexic responses to intravenous infusions of gut hormones peptide YY-(3-36) [PYY-(3-36)] and glucagon-like peptide-1 (GLP-1). Rats received three intraperitoneal injections of CAP or vehicle (VEH) in 24 h. After recovery, non-food-deprived rats received at dark onset a 3-h intravenous infusion of CCK-8 (5, 17 pmol·kg⁻¹·min⁻¹), PYY-(3-36) (5, 17, 50 pmol·kg⁻¹·min⁻¹), or GLP-1 (17, 50 pmol·kg⁻¹·min⁻¹). CCK-8 was much less effective in reducing food intake in CAP vs. VEH rats. CCK-8 at 5 and 17 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 39 and 71% in VEH rats and 7 and 18% in CAP rats. In contrast, PYY-(3-36) and GLP-1 were similarly effective in reducing food intake in VEH and CAP rats. PYY-(3-36) at 5, 17, and 50 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 15, 33, and 70% in VEH rats and 13, 30, and 33% in CAP rats. GLP-1 at 17 and 50 pmol·kg⁻¹·min⁻¹ reduced food intake during the 3-h infusion period by 48 and 60% in VEH rats and 30 and 52% in CAP rats. These results suggest that anorexic responses to PYY-(3-36) and GLP-1 are not primarily mediated by the CAP-sensitive peripheral sensory neurons (presumably vagal) that mediate CCK-8-induced anorexia.

  16. Ex Vivo Human Pancreatic Slice Preparations Offer a Valuable Model for Studying Pancreatic Exocrine Biology.

    PubMed

    Liang, Tao; Dolai, Subhankar; Xie, Li; Winter, Erin; Orabi, Abrahim I; Karimian, Negar; Cosen-Binker, Laura I; Huang, Ya-Chi; Thorn, Peter; Cattral, Mark; Gaisano, Herbert Y

    2017-02-27

    A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathological responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis.

  17. Preparation of tyrosine-O-(/sup 35/S)sulfated cholecystokinin octapeptide from a nonsulfated precursor peptide

    SciTech Connect

    Nakahara, T.; Waki, M.; Uchimura, H.; Hirano, M.; Kim, J.S.; Matsumoto, T.; Nakamura, K.; Ishibashi, K.; Hirano, H.; Shiraishi, A.

    1986-04-01

    A rapid and simple one-pot method for O-sulfation of nonsulfated cholecystokinin octapeptide (CCK-8) was developed using sulfuric acid and dicyclohexylcarbodiimide (DCC) without protection of the amino acid side chains. The extent of sulfation was increased with increasing the amount of reactants, sulfuric acid, and DCC, and reached maximum (40%) with fourfold molar excess of sulfuric acid and 40-fold molar excess of DCC. The excess of nonsulfated peptide inhibited the sulfation. The sulfation product was purified by HPLC or TLC to give a pure sulfated substance which showed exactly the same behavior as that of an authentic O-sulfated CCK-8 on HPLC or TLC. The purified sulfated peptide was active in stimulating amylase secretion from rat pancreatic fragments, and amino acid analysis showed that the tyrosine residue in the peptide existed in O-sulfated form. Sulfation with (/sup 35/S)sulfuric acid-DCC produced a radioactive substance, from which O-(/sup 35/S)sulfated CCK-8 could be easily purified by two-dimensional TLC.

  18. Cholecystokinin action on layer 6b neurons in somatosensory cortex

    PubMed Central

    Chung, Leeyup; Moore, Scott D.; Cox, Charles L.

    2009-01-01

    Layer 6b in neocortex is a distinct sublamina at the ventral portion of layer 6. Corticothalamic projections arise from 6b neurons, but few studies have examined the functional properties of these cells. In the present study we examined the actions of cholecystokinin (CCK) on layer 6b neocortical neurons using whole-cell patch clamp recording techniques. We found that the general CCK receptor agonist CCK8S (sulfated CCK octapeptide) strongly depolarized the neurons, and this action persisted in the presence of tetrodotoxin, suggesting a postsynaptic site of action. The excitatory actions of CCK8S were mimicked by the selective CCKB receptor agonist CCK4, and attenuated by the selective CCKB receptor antagonist L365260, indicating a role for CCKB receptors. Voltage clamp recordings revealed that CCK8S produced a slow inward current associated with a decreased conductance with a reversal potential near the K+ equilibrium potential. In addition, intracellular cesium also blocked the inward current, suggesting the involvement of a K+ conductance, likely Kleak. Our data indicate that CCK, acting via CCKB receptors, produces a long-lasting excitation of layer 6b neocortical neurons, and this action may play a critical role in modulation of corticothalamic circuit activity. PMID:19497313

  19. Interaction of apolipoprotein AIV with cholecystokinin on the control of food intake.

    PubMed

    Lo, Chun Min; Zhang, Dian Ming; Pearson, Kevin; Ma, Liyun; Sun, William; Sakai, Randall R; Davidson, W Sean; Liu, Min; Raybould, Helen E; Woods, Stephen C; Tso, Patrick

    2007-10-01

    Apolipoprotein AIV (apo AIV) and cholecystokinin (CCK) are peptides that act both peripherally and centrally to reduce food intake by decreasing meal size. The present study examined the effects of intraperitoneally administered bolus doses of recombinant apo AIV, CCK-8, and a combination of subthreshold doses of apo AIV and CCK on 4-h food intake in rats that were fasted overnight. Apo AIV at 100 microg/kg reduced food intake significantly relative to the saline control for 1 h, as did doses of CCK-8 at or above 0.125 microg/kg. Doses of apo AIV (50 microg/kg) or CCK (0.06 microg/kg) alone had no effect on food intake. However, when these subthreshold doses of apo AIV and CCK were administered together, the combination produced a significant inhibition of food intake relative to saline controls (P < 0.001), and the duration of the effect was longer than that caused by the administration of either apo AIV or CCK alone. The satiation effect produced by CCK-8 + apo AIV was attenuated by lorglumide, a CCK1 receptor antagonist. We conclude that, whereas the intraperitoneal administration of doses of either recombinant apo AIV or CCK at or above threshold levels reduces food intake, the coadministration of subthreshold doses of the two peptides is highly satiating and works via CCK1 receptor.

  20. Honokiol induces cell cycle arrest and apoptosis in human gastric carcinoma MGC-803 cell line.

    PubMed

    Yan, Bin; Peng, Zhi-Yong

    2015-01-01

    Gastric carcinoma is a malignant tumor that responds poorly to both chemotherapy and radiation therapy. In our study, we investigated the anti-cancer effect of honokiol, an active component isolated and purified from the Magnolia officinalis, in human gastric carcinoma MGC-803 cell line. The cell viability was detected by the CCK8 assay. The cell apoptosis and cell cycle arrest were assessed by flow cytometer. The protein expression of cell cycle regulators and tumor suppressors were analyzed by western blotting. Treatment of human gastric carcinoma cells with honokiol induced cell death in a dose-and time-dependent manner by using CCK8 assay. Consistent with the CCK8 assay, the flow cytometry results showed that the proportion of apoptosis cells had gained when the cells were exposed to honokiol. Moreover, Cyclin B1, CDC2 and cdc25C were downregulated, and the expression of p-CDC2 and p-cdc25c was significantly upregulated upon honokiol treatment. P53 and p21 were significantly upregulated by honokiol treatment. Treatment of MGC-803 cells with honokiol significantly increased the pro-apoptotic Bax level and decreased the anti-apoptotic Bcl-2 level. These results confirmed that honokiol could induce apoptosis and cell cycle arrest, the underlying molecular mechanisms, at least partially, through activation p53 signaling and downregulation CDC2/cdc25C expression.

  1. Pharmacological characterisation of a new potent and specific nonpolypeptidic cholecystokinin antagonist.

    PubMed

    Setnikar, I; Bani, M; Cereda, R; Chisté, R; Makovec, F; Pacini, M A; Revel, L; Rovati, L C; Rovati, L A

    1987-06-01

    D,L-4-(3,4-Dichloro-benzoylamino)-5-(N-3-methoxypropyl- pentylamino)-5-oxo-pentanoic acid (CR 1505) belongs to a newly discovered class of agents with cholecystokinin (CCK) antagonistic activity. CR 1505 displaces CCK-8 from the central CCK receptors at concentrations of 9.1 mumol/l, and from the peripheral CCK receptors at concentrations of 0.33 mumol/l. CR 1505 antagonizes in vitro the contractant effects of CCK-8 on gall bladder strips of the guinea pig at 0.79 mumol/l and those on the small intestine at 1.6 mumol/l. These antagonistic effects are dose dependent and of competitive type. The antagonistic activities of CR 1505 against contractions of smooth muscles elicited by CCK-8 are at least 1000 times more potent than those against the contractions elicited by acetylcholine, BaCl2, histamine, serotonin, Substance P, bradykinin or dimethylphenylpiperazine. CR 1505 is also practically ineffective against the contractions of the small intestine of the guinea pig elicited by electrical field stimulations either as "cholinergic twich" (0.05 Hz), or as "cholinergic contractions" (trains of 10 min at 1 Hz), or as "non-cholinergic contractions" (200 impulses at 5 Hz in presence of atropine). CR 1505 is therefore a potent, specific, competitive and reversible CCK antagonist.

  2. Duodenal myotomy blocks reduction of meal size and prolongation of intermeal interval by cholecystokinin.

    PubMed

    Lateef, Dalya M; Washington, Martha C; Raboin, Shannon J; Roberson, Allison E; Mansour, Mahmoud M; Williams, Carol S; Sayegh, Ayman I

    2012-02-01

    We have shown that vagotomy (VGX) attenuates the reduction of meal size (MS) produced by cholecystokinin (CCK) -8 and -33 and that celiaco-mesenteric ganglionectomy (CMGX) attenuates the prolongation of the intermeal interval (IMI) produced by CCK-33. Here, we report the following novel data. First, by determining the distribution of CCK(1) receptor messenger RNA, which mediates reduction of MS and prolongation of IMI by CCK, in seven regions of the gastrointestinal tract in the adult rat we found that the duodenum contains the highest concentration of this receptor in the gut. Second, based on the previous finding we performed a unique surgical technique known as duodenal myotomy (MYO), which severs all the nerves of the gut wall in the duodenum including vagus, splanchnic and enteric nerves. Third, we determined MS and IMI in duodenal MYO rats in responses to endogenous CCK-58 released by the non-nutrient, trypsin inhibitor, camostat and CCK-8 to test the possibility that the duodenum is the site of action for reduction of MS and prolongation of IMI. We found that, similar to the previous work reported by using CCK-8 and MS, duodenal MYO also blocked reduction of MS by camostat. Forth, duodenal MYO blocked prolongation of IMI by camostat. As such, our current results suggest that the duodenum is the gut site that communicates both feeding signals of endogenous CCK, MS and IMI, with the brain through vagal and splanchnic afferents.

  3. Enhanced Neurohypophyseal Vasopressin Release is Associated with Increased Opioid Inhibition of Oxytocin Release.

    PubMed

    Heijning, B J; Herik, I K; Rots, N Y; Greidanus, T B

    1991-02-01

    Abstract We tested the hypothesis of a cross-inhibition of oxytocin (OT) release by endogenous opioid peptides co-released with vasopressin (VP). This opioid cross-inhibition resulted in a selective block of OT release and hence in preferential release of VP. The effects of the opiate receptor antagonist naloxone were tested on neurohypophyseal VP release during dehydration, ethanol administration and sulphated cholecystokinin octapeptide (CCK-8S) application, assuming that the inhibition of pituitary OT release by endogenous opioids increases as neurohypophyseal VP output increases. A high VP output was found to coincide with increased inhibition of OT release: Subcutaneous injection of graded doses of naloxone (30 min prior to decapitation), augmented OT plasma levels significantly more in 24 h water-deprived male rats than in normally hydrated rats. Naloxone had no effect on VP release. Ethanol (10% in saline) administered intragastrically 50 min prior to decapitation and 20 min before subcutaneous naloxone (5 mg/kg) resulted in the inhibition of VP output. The ethanol treatment resulted in a rise in plasma OT levels that was additional to the effect of naloxone. These features were present in normally hydrated as well as in 24 h water-deprived animals, but were more pronounced in the latter group. Peripheral CCK-8S administration induces an abrupt and selective secretion of OT. Blocking the opioid inhibition of OT release with naloxone resulted in a significant rise of OT compared to that with CCK-8S alone. The magnitude of the opioid inhibition coincided with the activity of the VP system, and a higher dose of naloxone was needed to potentiate the CCK-8S effect on OT release in the water-deprived group than in euhydrated rats. No effect of CCK-8S and/or naloxone was found on VP plasma levels. The data indicate that opioid peptides co-released with VP (like dynorphin) may be responsible for cross-inhibition of OT release during dehydration. This suggests that

  4. Activation of Nesfatin-1-Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin-1 in Rats.

    PubMed

    Saito, R; So, M; Motojima, Y; Matsuura, T; Yoshimura, M; Hashimoto, H; Yamamoto, Y; Kusuhara, K; Ueta, Y

    2016-09-01

    Peripheral anorectic hormones, such as glucagon-like peptide (GLP)-1, cholecystokinin (CCK)-8 and leptin, suppress food intake. The newly-identified anorectic neuropeptide, nesfatin-1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP-1 and CCK-8 and co-administrations of GLP-1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin-1-immunoreactive (-IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP-1 (100 μg/kg) caused significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK-8 (50 μg/kg) resulted in marked increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP-1 (33 μg/kg) or leptin. However, co-administration of GLP-1 (33 μg/kg) and leptin resulted in significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP-1, CCK-8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin-1. These results indicate that nesfatin-1-expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP-1 and leptin in addition to CCK-8, and also suppress food intake in

  5. The effect of Korean pine nut oil on in vitro CCK release, on appetite sensations and on gut hormones in post-menopausal overweight women.

    PubMed

    Pasman, Wilrike J; Heimerikx, Jos; Rubingh, Carina M; van den Berg, Robin; O'Shea, Marianne; Gambelli, Luisa; Hendriks, Henk F J; Einerhand, Alexandra W C; Scott, Corey; Keizer, Hiskias G; Mennen, Louise I

    2008-03-20

    Appetite suppressants may be one strategy in the fight against obesity. This study evaluated whether Korean pine nut free fatty acids (FFA) and triglycerides (TG) work as an appetite suppressant. Korean pine nut FFA were evaluated in STC-1 cell culture for their ability to increase cholecystokinin (CCK-8) secretion vs. several other dietary fatty acids from Italian stone pine nut fatty acids, oleic acid, linoleic acid, alpha-linolenic acid, and capric acid used as a control. At 50 muM concentration, Korean pine nut FFA produced the greatest amount of CCK-8 release (493 pg/ml) relative to the other fatty acids and control (46 pg/ml). A randomized, placebo-controlled, double-blind cross-over trial including 18 overweight post-menopausal women was performed. Subjects received capsules with 3 g Korean pine (Pinus koraiensis) nut FFA, 3 g pine nut TG or 3 g placebo (olive oil) in combination with a light breakfast. At 0, 30, 60, 90, 120, 180 and 240 minutes the gut hormones cholecystokinin (CCK-8), glucagon like peptide-1 (GLP-1), peptide YY (PYY) and ghrelin, and appetite sensations were measured. A wash-out period of one week separated each intervention day.CCK-8 was higher 30 min after pine nut FFA and 60 min after pine nut TG when compared to placebo (p < 0.01). GLP-1 was higher 60 min after pine nut FFA compared to placebo (p < 0.01). Over a period of 4 hours the total amount of plasma CCK-8 was 60% higher after pine nut FFA and 22% higher after pine nut TG than after placebo (p < 0.01). For GLP-1 this difference was 25% after pine nut FFA (P < 0.05). Ghrelin and PYY levels were not different between groups. The appetite sensation "prospective food intake" was 36% lower after pine nut FFA relative to placebo (P < 0.05). This study suggests that Korean pine nut may work as an appetite suppressant through an increasing effect on satiety hormones and a reduced prospective food intake.

  6. An immunocytochemical and ultrastructural study of the larval anterior intestine of the frog Rana temporaria, with especial reference to endocrine cells.

    PubMed

    Bodegas, M E; Villaro, A C; Burrell, M A; Rovira, J; Valverde, E; Ortiz De Zárate, A; Sesma, P

    1997-10-01

    Endocrine cells of the larval intestine of Rana temporaria tadpoles have been identified by argyrophilic, immunocytochemical and electron-microscopical techniques. Scarce endocrine cells have been found in both the short non-absorptive zone immediately following the stomach, and in the rest of the anterior intestine. Endocrine cells are frequently seen to extend a cytoplasmic process towards the lumen. Immunoreactivity for serotonin, somatostatin, bombesin and cholecystokinin-8 has been detected. According to the ultrastructural traits of the endocrine granules, three larval intestinal endocrine populations have been differentiated.

  7. Unexpected side products in the conjugation of an amine-derivatized morpholino oligomer with p-isothiocyanate benzyl DTPA and their removal.

    PubMed

    Liu, Guozheng; Dou, Shuping; Liu, Yuxia; Liang, Minmin; Chen, Ling; Cheng, Dengfeng; Greiner, Dale; Rusckowski, Mary; Hnatowich, Donald J

    2011-02-01

    In connection with pretargeting, an amine-derivatized morpholino phosphorodiamidate oligomer (NH(2)-cMORF) was conjugated conventionally with p-isothiocyanate benzyl-DTPA (p-SCN-Bn-DTPA). However, after (111)In radiolabeling, unexpected label instability was observed. To understand this instability, the NH(2)-cMORF and, as control, the native cMORF without the amine were conjugated in the conventional manner. Surprisingly, the (111)In labeling of the native cMORF conjugate was equally effective as that of the NH(2)-cMORF conjugate (>95%) despite the absence of the amine group. Furthermore, heating the radiolabeled NH(2)-cMORF and native cMORF conjugates resulted in a 35% loss and a complete loss of the label, respectively. Since the (111)In labeled DTPA is known to be stable, the instability in both cases must be due to some unstable association of DTPA to the cMORF, presumably unstable association to some endogenous sites in cMORF. Based on this assumption, a postconjugation-prepurification heating step was introduced, and labeling efficiency and stability were again investigated. By introducing the heating step, the side products were dissociated, and after purification and labeling, the NH(2)-cMORF conjugate provided a stable label and high labeling efficiency with no need for postlabeling purification. The biodistribution of this radiolabeled conjugate in normal mice showed significantly lower backgrounds compared with the labeled unstable native cMORF conjugate. In conclusion, the conventional conjugation procedure to attach the p-SCN-Bn-DTPA to NH(2)-cMORF resulted in side product(s) that were responsible for the (111)In label instability. Adding a postconjugation-prepurification heating step dissociated the side products, improved the label stability and lowered tissue backgrounds in mice. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Development of PEGylated carboxylic acid-modified polyamidoamine dendrimers as bone-targeting carriers for the treatment of bone diseases.

    PubMed

    Yamashita, Shugo; Katsumi, Hidemasa; Hibino, Nozomi; Isobe, Yugo; Yagi, Yumiko; Kusamori, Kosuke; Sakane, Toshiyasu; Yamamoto, Akira

    2017-09-28

    In this study, we aimed to develop a polyethylene glycol (PEG)-conjugated third generation polyamidoamine (PAMAM) dendrimer with multiple carboxylic acids as a bone-targeting carrier for the treatment of bone diseases. We conjugated PAMAM backbones to various carboxylic acids [aspartic acid (Asp), glutamic acid (Glu), succinic acid (Suc), or aconitic acid (Aco)] to obtain four different types of carboxylic acid-modified PAMAMs. PEG was covalently bound to carboxylic acid-modified PAMAMs to obtain PEGylated carboxylic acid-modified PAMAMs. In a tissue distribution study, the amount of (111)In-labeled unmodified PAMAM taken up by the bone after intravenous injection in mice was 11.3%. In contrast, the dose of (111)In-labeled PEG(5)-Asp-PAMAM, PEG(5)-Glu-PAMAM, PEG(5)-Suc-PAMAM, or PEG(5)-Aco-PAMAM that accumulated in the bone after injection was approximately 46.0, 15.6, 22.6, and 24.5%, respectively. The bone clearance rates of (111)In-labeled PEGylated carboxylic acid-modified PAMAMs were proportional to their affinities to hydroxyapatite and Ca(2+). An intra-bone distribution study showed that fluorescein isothiocyanate-labeled PEG(5)-Asp-PAMAM predominantly accumulated on eroded and quiescent surfaces, a pattern associated with the pathogenesis of bone diseases, such as rheumatoid arthritis and osteoporosis. Our findings indicate that PEG(5)-Asp-PAMAM is a promising drug carrier for efficient drug targeting to the bones. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons

    SciTech Connect

    Savage, B.; McFadden, P.R.; Hanson, S.R.; Harker, L.A.

    1986-08-01

    The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In-labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high-density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6).

  10. Donor-leukocyte imaging in granulocytopenic patients with suspected abscesses: concise communication

    SciTech Connect

    Anstall, H.B.; Coleman, R.E.

    1982-04-01

    /sup 111/In-labeled donor leukocytes were used for the detection of foci of suppuration in eight severely leukopenic patients with marrow suppression, either idiopathic or associated with chemotherapeutic regimens for leukemia. In three patients good correlation was found between the results of imaging and clinical signs or subsequent proof of inflammation. In the other five patients, in whom no evidence of localized suppuration occurred, no abnormal accumulations of radioactivity were demonstrable. Labeled donor leukocytes provide a method for locating suppurative foci in severely leukopenic patients in whom autologous leukocyte labeling is impractical.

  11. Comparison of three remote radiolabelling methods for long-circulating liposomes.

    PubMed

    van der Geest, Tessa; Laverman, Peter; Gerrits, Danny; Franssen, Gerben M; Metselaar, Josbert M; Storm, Gert; Boerman, Otto C

    2015-12-28

    Long-circulating liposomes (LCL) are often used as a drug carrier system to improve the therapeutic index of water-soluble drugs. To track these LCL in vivo, they can be radiolabelled with (111)In-oxine. For this labelling method, generally DTPA is encapsulated in the aqueous phase of LCL (DTPA-LCL). Alternatively, LCL can be labelled with (111)InCl3 after incorporation of DTPA-conjugated DSPE in the lipid bilayer (DTPA-DSPE LCL). Here, we compared the in vitro properties of DTPA-DSPE LCL with those of DTPA LCL and empty LCL. Additionally, we compared the in vivo performance of DTPA-DSPE LCL with those of DTPA LCL in mice. DTPA LCL (88 nm) and empty LCL (84 nm) were labelled with (111)In-oxine, and DTPA-DSPE LCL (83 nm) were labelled with (111)InCl3. Labelling efficiency at increasing specific activity was determined. In vitro stability of (111)In-labelled LCL was determined in human serum at 37 °C. The in vivo properties of (111)In-labelled LCL were determined in mice with a Staphylococcus aureus infection in the thigh muscle. Image acquisition, blood sampling and biodistribution studies were performed 1, 4 (blood sampling only), 24, 48 and 72 h p.i. of (111)In-labelled LCL. DTPA-DSPE LCL could be labelled efficiently at a much higher specific activity compared to DTPA LCL and empty LCL: > 90% at 15 GBq/mmol, > 90% at 150 MBq/mmol and 60–65% at 150 MBq/mmol, respectively. (111)In-labelled DTPA-DSPE LCL and DTPA LCL were stable in human serum, regarding label retention, for at least 48 h at 37 °C (> 98% retention of the radiolabel). In contrast, only 68% radiolabel was retained in empty LCL after 48 h. In vivo targeting of (111)In-DTPA-DSPE LCL to the abscess was comparable to targeting of (111)In-DTPA LCL (3.5 ± 0.9%ID/g and 3.4 ± 0.9%ID/g abscess uptake respectively, 48 h p.i.). In conclusion, labelling of DTPA-DSPE LCL with (111)InCl3 represents a robust, easy and fast procedure which is preferred over the more laborious conventional labelling of DTPA

  12. Early diagnosis of acute postoperative renal transplant rejection by indium-111-labeled platelet scintigraphy

    SciTech Connect

    Tisdale, P.L.; Collier, B.D.; Kauffman, H.M.; Adams, M.B.; Isitman, A.T.; Hellman, R.S.; Hoffmann, R.G.; Rao, S.A.; Joestgen, T.; Krohn, L.

    1986-08-01

    A prospective evaluation of /sup 111/In-labeled platelet scintigraphy (IPS) for the early diagnosis of acute postoperative renal transplant rejection (TR) was undertaken. The results of IPS were compared with in vitro biochemical tests, the clinical finding of graft tenderness, and combined (/sup 99m/Tc)DTPA and (/sup 131/I)orthoiodohippurate scintigraphy. With a sensitivity of 0.93 and a specificity of 0.95, IPS provided otherwise unavailable diagnostic information. Furthermore, postoperative IPS was a good predictor of long-term allograft survival.

  13. Radionuclide evaluation of renal transplants

    SciTech Connect

    Dubovsky, E.V.; Russell, C.D.

    1988-07-01

    In this review article, the following topics are treated: the radiopharmaceuticals /sup 99m/Tc-diethylenetriaminepentaacetic acid (DTPA), /sup 131/I-orthoiodohippurate (OIH), /sup 99m/Tc-mercaptoacetyltriglycine (MAG3), /sup 67/Ga-citrate, radioiodinated fibrinogen, /sup 99m/Tc-sulfur colloid, 111In-labelled white cells and platelets; gamma camera methods based on images, on first pass and on tubular transit; blood clearance methods; and the diagnosis of surgical complications, acute rejection (AR), acute tubular necrosis (ATN), chronic rejection (CR), and cyclosporine-A (CYA) toxicity. 94 references.

  14. [Experimental study of indium-111 labeled octreotide application for radionuclide diagnostics of tumors with high expression of somatostatin receptors].

    PubMed

    Grinin, M G; Shimanovskiĭ, N L

    2011-01-01

    We have studied the possibility of using octreoitid labeled by indium-111 for radionuclide diagnostics of neoplasms with high expression of somatostatin receptors. It has been established that, using experiments with cell cultures, it is possible to predict the application of 111In-labeled octreotid and determine time intervals for the best visualization of tumors. Indium-111-labeled octreotid accumulation in animals with transferred B16 tumor (pigment melanoma) shows evidence for the opportunity of using this substance as radiopharmaceutical medium for tumor diagnostics.

  15. Noninvasive radioisotopic technique for detection of platelet deposition on bovine pericardial mitral-valve prosthesis and in vitro quantification of visceral microembolism in dogs

    SciTech Connect

    Dewanjee, M.K.; Trastek, V.F.; Tago, M.; Torianni, M.; Kaye, M.P.

    1983-01-01

    Platelet deposition on bovine pericardial-tissue mitral-valve prostheses in dogs was observed noninvasively by use of /sup 111/In-labeled platelets and quantified after sacrifice at one, 14 and 30 days postimplantation (300-400 microCi of labeled platelets having been injected 24 hrs previously). Thrombosis on the sewing ring and pericardial leaflets at one and 14 days and on the leaflets at 30 days was delineated in scintiphotos. In vitro quantification (% injected dose) indicated that the sewing ring and perivalvular tissue retained 0.75% of labeled platelets at one day postimplantation, 0.084% at 14 days, and 0.0042% at 30 days. Platelet survival was reduced to 38 hrs at 21 days postimplantation but returned toward the normal (50 hrs) with endothelial covering of the sewing ring. Microemboli in lung and kidney, as measured by tissue/blood radioactivity ratio, decreased significantly at 30 days. /sup 111/In-labeled platelets thus provide a sensitive marker for noninvasive imaging and in vitro quantification of platelet deposition on valvular prostheses and microemboli trapped in viscera.

  16. Alloimmunization prevents the migration of transfused indium-111-labeled granulocytes to sites of infection

    SciTech Connect

    Dutcher, J.P.; Schiffer, C.A.; Johnston, G.S.; Papenburg, D.; Daly, P.A.; Aisner, J.; Wiernik, P.H.

    1983-08-01

    111In-labeled granulocytes were used to study the effects of histocompatibility factors on the migration of transfused granulocytes to infected sites. Fourteen alloimmunized and 20 nonalloimmunized patients received approximately 10(8) 111In-labeled granulocytes from ABO-compatible, non-HLA-matched donors, and scans were performed over known infected sites. All 14 alloimmunized patients had lymphocytotoxic antibody (LCTAb) and required HLA-matched platelet transfusions. Of the nonalloimmunized patients, 20/20 had positive scans at sites of infection. None of the 20 had LCTAb, 0/17 had a positive lymphocytotoxic crossmatch (LCTXM) with the donor, and 3/18 had a positive leukoagglutinin crossmatch (LAXM). Thus, histocompatibility testing was not found to be important in nonalloimmunized patients. In contrast, only 3/14 alloimmunized patients had positive scans at sites of infection (p . 0.00001 compared to nonalloimmunized patients). One of 3 had a positive LCTXM and 2/3 had a positive LAXM. Of the alloimmunized patients, 10/11 with negative scans had a positive LCTXM and 8/11 had a positive LAXM. Labeled granulocytes failed to reach sites of infection in 11/14 (78%) alloimmunized patients, demonstrating that histocompatibility factors can be of major importance in affecting the outcome of granulocyte transfusions. Granulocytes from random donors are unlikely to be effective in alloimmunized patients. The lack of an adequate crossmatching technique is a major problem limiting the ability to provide granulocyte transfusions for alloimmunized patients.

  17. Development of a new radiolabel (lead-203) and new chelating agents for labeling monoclonal anntibodies for imaging

    SciTech Connect

    Srivastava, S.C.; Mease, R.C.; Meinken, G.E.; Mausner, L.F.; Steplewski, Z.

    1988-01-01

    High liver uptake and slow body clearance presently limit the usefulness of /sup 111/In labeled antibodies for tumor imaging. We have investigated /sup 203/Pb as an alternate and better antibody label. The DTPA and cyclohexyl EDTA (CDTA) conjugates of an anticolon carcinoma antibody, 17-1A were labeled (bicyclic anhydride method) with /sup 203/Pb and /sup 111/In with 60 and 90% labeling yields, respectively. The biodistribution of /sup 203/Pb-17-1A conjugates was compared with the corresponding /sup 111/In-labeled preparations and with /sup 203/Pb-DTPA, /sup 203/Pb-nitrate and nonrelevant antibody controls in normal and human tumor (SW948) xenografted nude mice at 24, and 96 hr. Lead-203-labeled CDTA and DTPA antibody conjugates gave similar in vivo distributions. Even though the lead bound to these chelate-antibody conjugates was more labile in serum and in vivo, compared to indium, it cleared much faster from the liver and the whole body. A new series of chelating agents based on the incorporation of a trans-1,2- diaminocyclohexane moiety into the carbon backbone of polyaminocarboxylates is being synthesized. These are expected to provide stronger complexing ability for lead and produce greater in vivo stability. These ligands are also expected to be superior to EDTA and DTPA for labeling antibodies with other radiometals, including indium. 32 refs., 3 tabs.

  18. In vivo stability of ester- and ether-linked phospholipid-containing liposomes as measured by perturbed angular correlation spectroscopy.

    PubMed Central

    Derksen, J T; Baldeschwieler, J D; Scherphof, G L

    1988-01-01

    To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing 111In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of 111In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor [G22(infinity)] of the 111In label. We found that MLVs but not SUVs, having DHPC as their bulk phospholipid, showed an increased resistance against lysosomal degradation as compared to other phospholipid-containing liposomes. The use of diacyl phospholipids with a high gel/liquid-crystalline phase-transition temperature, such as DPPC and DSPC, also retarded degradation of MLV, but not of SUV in the dose range tested, while the rate of uptake of these liposomes by the liver was lower. PMID:3200855

  19. Fate of gamma-interferon-activated killer blood monocytes adoptively transferred into the abdominal cavity of patients with peritoneal carcinomatosis

    SciTech Connect

    Stevenson, H.C.; Keenan, A.M.; Woodhouse, C.; Ottow, R.T.; Miller, P.; Steller, E.P.; Foon, K.A.; Abrams, P.G.; Beman, J.; Larson, S.M.

    1987-11-15

    Five patients with colorectal cancer widely metastatic to peritoneal surfaces have been treated i.p. with infusions of autologous blood monocytes made cytotoxic by in vitro incubation with human gamma-interferon. The monocytes were purified by a combination of cytapheresis and counter-current centrifugal elutriation procedures; each week approximately 350 million activated monocytes were given to patients as adoptive immunotherapy by a single i.p. instillation. On the eighth cycle of treatment the trafficking of i.p. infused blood monocytes was studied in two patients by prelabeling the cells with /sup 111/In. These activated cells became distributed widely within the peritoneal cavity. Two and 5 days after infusion their position within the peritoneum had not changed. When peritoneal specimens were obtained 36 h after /sup 111/In-labeled monocyte infusion, labeled monocytes were demonstrated to be associated with the serosal surfaces by autoradiographic analysis. Scintiscanning structures outside the abdominal cavity revealed that /sup 111/In-labeled monocytes infused i.p. did not traffic to other organs during the 5 days of the study. We conclude that i.p. adoptive transfer of autologous killer blood monocytes is an effective way of delivering these cytotoxic cells to sites of tumor burden on peritoneal surfaces in these cancer patients.

  20. Skeletal muscle satellite cell migration to injured tissue measured with 111In-oxine and high-resolution SPECT imaging

    PubMed Central

    Elster, Jennifer L.; Rathbone, Christopher R.; Liu, Zhonglin; Liu, Xiasong; Barrett, Harrison H.; Rhoads, Robert P.; Allen, Ronald E.

    2014-01-01

    The delivery of adult skeletal muscle stem cells, called satellite cells, to several injured muscles via the circulation would be useful, however, an improved understanding of cell fate and biodistribution following their delivery is important for this goal to be achieved. The objective of this study was to evaluate the ability of systemically delivered satellite cells to home to injured skeletal muscle using single-photon emission computed tomography (SPECT) imaging of 111In-labeled satellite cells. Satellite cells labeled with 111In-oxine and green fluorescent protein (GFP) were injected intravenously after bupivicaine-induced injury to the tibialis anterior muscle. Animals were imaged with a high-resolution SPECT system called FastSPECT II for up to 7 days after transplantation. In vivo FastSPECT II imaging demonstrated a three to five-fold greater number of transplanted satellite cells in bupivicaine-injured muscle as compared to un-injured muscle after transplantation; a finding that was verified through autoradiograph analysis and quantification of GFP expression. Satellite cells also accumulated in other organs including the lung, liver, and spleen, as determined by biodistribution measurements. These data support the ability of satellite cells to home to injured muscle and support the use of SPECT and autoradiograph imaging techniques to track systemically transplanted 111In labeled satellite cells in vivo, and suggest their homing may be improved by reducing their entrapment in filter organs. PMID:24190365

  1. Low density lipoprotein receptor-independent hepatic uptake of a synthetic, cholesterol-scavenging lipoprotein: implications for the treatment of receptor-deficient atherosclerosis

    SciTech Connect

    Williams, K.J.; Vallabhajosula, S.; Rahman, I.U.; Donnelly, T.M.; Parker, T.S.; Weinrauch, M.; Goldsmith, S.J.

    1988-01-01

    The metabolism of infused /sup 111/In-labeled phospholipid liposomes was examined in Watanabe heritable hyperlipidemic (WHHL) rabbits, which lack low density lipoprotein (LDL) receptors, and in normal control rabbits. The half-times (t/sub 1/2/) for clearance of /sup 111/In and excess phospholipid from plasma were 20.8 +/- 0.9 hr and 20.3 +/- 4.6 hr in WHHL and 20.0 +/- 0.8 hr and 19.6 +/- 2.2 hr in the normal rabbits. By 6 hr postinfusion, the plasma concentration of unesterified cholesterol increased by 2.2 +/- 0.23 mmol/liter in WHHL and 2.1 +/- 0.04 mmol/liter in normal rabbits, presumably reflecting mobilization of tissue sores. Disappearance of excess plasma cholesterol was > 90% complete in both groups of rabbits by 70 hr postinfusion. By quantitative ..gamma.. camera imaging, hepatic trapping of /sup 111/In-labeled liposomes over time was indistinguishable between the two groups. At autopsy, the liver was the major organ of clearance. Aortic uptake of /sup 111/In was < 0.02%. Thus, mobilization of cholesterol and hepatic uptake of phospholipid liposomes do not require LDL receptors. Because phospholipid infusions produce rapid substantial regression of atherosclerosis in genetically normal animals, the results suggest that phospholipid liposomes or triglyceride phospholipid emulsions (e.g., Intralipid) might reduce atherosclerosis in WHHL rabbits and in humans with familial hypercholesterolemia.

  2. Intracavitary use of two radiolabeled tumor-associated monoclonal antibodies

    SciTech Connect

    Malamitsi, J.; Skarlos, D.; Fotiou, S.; Papakostas, P.; Aravantinos, G.; Vassilarou, D.; Taylor-Papadimitriou, J.; Koutoulidis, K.; Hooker, G.; Snook, D.

    1988-12-01

    Six patients with metastatic breast cancer and malignant pleural effusions and 13 patients with known or suspected ovarian cancer, underwent immunoscintigraphy after intracavitary (intrapleural or intraperitoneal) administration of iodine-131-(131I) or indium-111-(111In) labeled tumor associated monoclonal antibodies HMFG2 and H17E2. This method proved to be sensitive and specific with a true-positive result in 13 out of 14 patients with tumor and a true-negative result in five out of five patients without tumor. At any one time, 65%-80% of the whole-body radioactivity was closely associated with the cavity into which the radiolabeled antibody was administered while the radioactivity in the blood was always low, (approximately 4 X 10(-3) of administered dose/ml of blood). Concentrations of radiolabeled antibody (per gram of tumor tissue) ranged from 0.02%-0.1% of the injected dose in intracavitary tumors, but only 0.002% in a retroperitoneal metastasis. The specificity of this approach was documented in four control patients with benign ovarian cysts and in two patients who were imaged using both specific and nonspecific radiolabeled antibody. We conclude that the intracavitary administration of 131I- or 111In-labeled HMFG2 and H17E2 is a favorable route of administration and offers significant advantages over previously reported intravenous administration for the localization of breast or ovarian metastases confined to the pleural or peritoneal cavities.

  3. Methods of assessment of thrombosis in vivo

    SciTech Connect

    Dewanjee, M.K.

    1987-01-01

    The contributions of platelets and clotting factors in thrombosis on injured vessel and cardiovascular prostheses have been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and humans with /sup 111/In-labeled platelets, /sup 123/I- and /sup 131/I-labeled fibrinogen, /sup 111/In-labeled antibody to the fibrinogen receptor on the platelet membrane and to fibrin. Thrombus localization by imaging was possible for large thrombus in vessel with deep injury of thrombogenic surface in the acute phase. A single layer of adherent platelets could not be imaged, due to the high background radioactivity present in blood. Thrombogenicity of grafts was compared with that of contralateral vessel. The dynamic process of platelet deposition could be followed accurately using the in vivo imaging technique. In addition, in vitro quantification permits determination of platelet and fibrin density and of the number of fibrin monomers per platelet in thrombus. The roles of prostacyclin, thromboxane inhibitors, and nonsteroidal antiinflammatory drugs have also been evaluated in animals models and humans. The tracer techniques thus provide invaluable information about platelet-fibrin deposition, its organization and dissolution, and for development of less thrombogenic surfaces for use in cardiovascular prostheses. 53 references.

  4. Noninvasive assessment of treatment of cardiac allograft rejection with indium-111-labeled lymphocytes

    SciTech Connect

    Rosenbloom, M.; Eisen, H.J.; Laschinger, J.; Saffitz, J.E.; Sobel, B.E.; Bergmann, S.R.; Bolman, R.M. III

    1988-09-01

    We have shown previously that cardiac allograft rejection can be detected noninvasively with gamma scintigraphy after administration of indium-111 (111In)-labeled lymphocytes. To determine whether this technique could be used to monitor salvage immunosuppressive therapy in reversing rejection, 5 dogs were studied after thoracic heterotopic cardiac transplantation. Initial postoperative immunosuppression was maintained with cyclosporine (10-20 mg/kg/day) and prednisone (1 mg/kg/day) for 7 days after transplantation and then discontinued. Scintigraphy after administration of labeled lymphocytes was performed during initial immunosuppression and every 3 days after its termination. Endomyocardial biopsies were obtained on each day scintigraphy was performed. Once scintigraphic criteria for rejection were met (111In-lymphocyte uptake greater than mean +/- 2SD of normal myocardium), animals were treated with high dose methylprednisolone and cyclosporine. Myocardial 111In-lymphocyte activity compared with that in blood was 0.7 +/- 0.8 during initial immunosuppression, increased to 5.7 +/- 3.5 after termination of therapy (P less than 0.01), and diminished with salvage immunosuppressive therapy to 0.5 +/- 0.8 (P = NS compared with native hearts or allografts during initial immunosuppression). Scintigraphy accurately predicted all but one episode of biopsy-documented rejection and accurately detected reversal of rejection during salvage. Thus, scintigraphy with 111In-labeled lymphocytes should facilitate noninvasive monitoring of antirejection therapy in patients.

  5. Comparison of the effects of dietary sunflower oil and virgin olive oil on rat exocrine pancreatic secretion in vivo.

    PubMed

    Díaz, Ricardo J; Yago, María D; Martínez-Victoria, Emilio; Naranjo, José A; Martínez, María A; Mañas, Mariano

    2003-11-01

    The aim of this study was to investigate the functional consequences in vivo of adapting the rat exocrine pancreas to different dietary fats. Weanling rats were fed diets containing 10 wt% virgin olive oil or sunflower oil for 8 wk. We then examined resting and cholecystokinin-octapeptide (CCK-8)-stimulated pancreatic secretion in the anesthetized animals. To confirm a direct influence of the type of fat upon the gland, the FA composition of pancreatic membranes as well as tissue protein and amylase content were determined in separate rats. The membrane FA profile was profoundly altered by the diets, reflecting the type of dietary fat given, although this was not paralleled by variations in the pancreatic content of protein or amylase. Nevertheless, dietary intake of oils evoked different effects on in vivo secretory activity. Resting flow rate and amylase output were significantly (P < 0.05) enhanced by sunflower oil feeding. Time course changes in response to CCK-8 infusion also showed a different pattern in each group. Secretion of fluid, protein, and amylase increased markedly in all animals, reaching a maximum within 20-40 min of infusion that was followed by a dramatic decline in both groups. In the sunflower oil group, this resulted in values reaching the resting level as soon as 60 min after CCK-8 infusion was begun. However, after the initial decline, olive oil group values showed a prolonged plateau elevation above the baseline (P < 0.05) that was maintained for at least the infusion time. In addition, a positive correlation between flow rate and both protein concentration and amylase activity existed in the olive oil group, but not in the sunflower oil group. The precise mechanism by which these effects are produced remains to be elucidated.

  6. Rat pancreas secretes particulate ecto-nucleotidase CD39

    PubMed Central

    Sørensen, Christiane E; Amstrup, Jan; Rasmussen, Hans N; Ankorina-Stark, Ieva; Novak, Ivana

    2003-01-01

    In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar-ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT-PCR and Western blotting we show that pancreas expresses the full length ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization on basolateral membranes of acini and intracellularly. In small intercalated/ interlobular ducts, CD39 immunofluorescence was localized on the luminal membranes, while in larger ducts it was localized on the basolateral membranes. Upon stimulation with cholecystokinin-octapeptide-8 (CCK-8), acinar CD39 relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK-8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti-CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic juice. This CD39 was confined only to the particulate and not to the soluble fraction of CCK-8-stimulated secretion. No CD39 activity was detected in secretion stimulated by secretin. The role of secreted particulate, possibly microsomal, CD39 would be to regulate intraluminal ATP concentrations within the ductal tree. In conclusion, we show a novel inducible release of full length particulate CD39, and propose its role in the physiological context of pancreatic secretion. PMID:12832497

  7. Roles of sphincter of Oddi motility and serum vasoactive intestinal peptide, gastrin and cholecystokinin octapeptide

    PubMed Central

    Zhang, Zhen-Hai; Qin, Cheng-Kun; Wu, Shuo-Dong; Xu, Jian; Cui, Xian-Ping; Wang, Zhi-Yi; Xian, Guo-Zhe

    2014-01-01

    AIM: To investigate roles of sphincter of Oddi (SO) motility played in pigment gallbladder stone formation in model of guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups: the control group and pigment stone group. The pigment stone group was divided into 4 subgroups with 6 guinea pigs each according to time of sacrifice, and were fed a pigment lithogenic diet and sacrificed after 3, 6, 9 and 12 wk. SO manometry and recording of myoelectric activity of the guinea pigs were obtained by multifunctional physiograph at each stage. Serum vasoactive intestinal peptide (VIP), gastrin and cholecystokinin octapeptide (CCK-8) were detected at each stage in the process of pigment gallbladder stone formation by enzyme-linked immunosorbent assay. RESULTS: The incidence of pigment gallstone formation was 0%, 0%, 16.7% and 66.7% in the 3-, 6-, 9- and 12-wk group, respectively. The frequency of myoelectric activity decreased in the 3-wk group. The amplitude of myoelectric activity had a tendency to decrease but not significantly. The frequency of the SO decreased significantly in the 9-wk group. The SO basal pressure and common bile duct pressure increased in the 12-wk group (25.19 ± 7.77 mmHg vs 40.56 ± 11.81 mmHg, 22.35 ± 7.60 mmHg vs 38.51 ± 11.57 mmHg, P < 0.05). Serum VIP was significantly elevated in the 6- and 12-wk groups and serum CCK-8 was decreased significantly in the 12-wk group. CONCLUSION: Pigment gallstone-causing diet may induce SO dysfunction. The tension of the SO increased. The disturbance in SO motility may play a role in pigment gallstone formation, and changes in serum VIP and CCK-8 may be important causes of SO dysfunction. PMID:24782626

  8. Evaluation of the effects of Cimicifugae Rhizoma on the morphology and viability of mesenchymal stem cells

    PubMed Central

    JEONG, SU-HYEON; LEE, JI-EUN; KIM, BO-BAE; KO, YOUNGKYUNG; PARK, JUN-BEOM

    2015-01-01

    Cimicifugae Rhizoma is a traditional herbal medicine used to treat various diseases in Korea, China and Japan. Cimicifugae Rhizoma is primarily derived from Cimicifuga heracleifolia Komarov or Cimicifuga foetida Linnaeus. Cimicifugae Rhizoma has been used as an anti-inflammatory, analgesic and antipyretic remedy. The present study was performed to evaluate the extracts of Cimicifugae Rhizoma on the morphology and viability of human stem cells derived from gingiva. Stem cells derived from gingiva were grown in the presence of Cimicifugae Rhizoma at final concentrations that ranged from 0.001 to 1,000 µg/ml. The morphology of the cells was viewed under an inverted microscope and the analysis of cell proliferation was performed using a Cell Counting kit-8 (CCK-8) assay on days 1, 3, 5 and 7. Under an optical microscope, the control cells exhibited a spindle-shaped, fibroblast-like morphology. The shapes of the cells in the groups treated with 0.001, 0.01, 0.1, 1 and 10 µg/ml Cimicifugae Rhizoma were similar to the shapes in the control group. Significant alterations in morphology were noted in the 100 and 1,000 µg/ml groups when compared with the control group. The cells in the 100 and 1,000 µg/ml groups were rounder, and fewer cells were present. The cultures that were grown in the presence of Cimicifugae Rhizoma at a concentration of 0.001 µg/ml on day 1 had an increased CCK-8 value. The cultures grown in the presence of Cimicifugae Rhizoma at a concentration of 10 µg/ml on day 7 had a reduced CCK-8 value. Within the limits of this study, Cimicifugae Rhizoma influenced the viability of stem cells derived from the gingiva, and its direct application onto oral tissues may have adverse effects at high concentrations. The concentration and application time of Cimicifugae Rhizoma should be meticulously controlled to obtain optimal results. PMID:26622366

  9. Cyclic cholecystokinin analogues with high selectivity for central receptors.

    PubMed Central

    Charpentier, B; Pelaprat, D; Durieux, C; Dor, A; Reibaud, M; Blanchard, J C; Roques, B P

    1988-01-01

    Taking as a model the N-terminal folding of the cholecystokinin tyrosine-sulfated octapeptide [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] deduced from conformational studies, two cyclic cholecystokinin (CCK) analogues were synthesized by conventional peptide synthesis: Boc-D-Asp-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 [compound I (Ahx, 2-aminohexanoic acid)] and Boc-gamma-D-Glu-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 (compound II). The binding characteristics of these peptides were investigated on brain cortex membranes and pancreatic acini of guinea pig. Compounds I and II were competitive inhibitors of [3H]Boc[Ahx28,31]CCK-(27-33) binding to central CCK receptors and showed a high degree of selectivity for these binding sites (compound I: Ki for pancreas/Ki for brain, 179; compound II: Ki for pancreas/Ki for brain, 1979). This high selectivity was associated with a high affinity for central CCK receptors (compound I: Ki, 5.1 nM; compound II: Ki, 0.49 nM). Similar affinities and selectivities were found when 125I Bolton-Hunter-labeled CCK-8 was used as a ligand. Moreover, these compounds were only weakly active in the stimulation of amylase release from guinea pig pancreatic acini (EC50 greater than 10,000 nM) and were unable to induce contractions in the guinea pig ileum (to 10(-6) M). The two cyclic CCK analogues, therefore, appear to be synthetic ligands exhibiting both high affinity and high selectivity for central CCK binding sites. These compounds could help clarify the respective role of central and peripheral receptors for various CCK-8-induced pharmacological effects. PMID:3162318

  10. High-protein diet improves sensitivity to cholecystokinin and shifts the cecal microbiome without altering brain inflammation in diet-induced obesity in rats.

    PubMed

    Wang, Lixin; Jacobs, Jonathan P; Lagishetty, Venu; Yuan, Pu-Qing; Wu, Shuping V; Million, Mulugeta; Reeve, Joseph R; Pisegna, Joseph R; Taché, Yvette

    2017-10-01

    High-protein diet (HPD) curtails obesity and/or fat mass, but it is unknown whether it reverses neuroinflammation or alters glucose levels, CCK sensitivity, and gut microbiome in rats fed a Western diet (WD)-induced obesity (DIO). Male rats fed a WD (high fat and sugar) for 12 wk were switched to a HPD for 6 wk. Body composition, food intake, meal pattern, sensitivity to intraperitoneal CCK-8S, blood glucose, brain signaling, and cecal microbiota were assessed. When compared with a normal diet, WD increased body weight (9.3%) and fat mass (73.4%). CCK-8S (1.8 or 5.2 nmol/kg) did not alter food intake and meal pattern in DIO rats. Switching to a HPD for 6 wk reduced fat mass (15.7%) with a nonsignificantly reduced body weight gain, normalized blood glucose, and decreased feeding after CCK-8S. DIO rats on the WD or switched to a HPD showed comparable microbial diversity. However, in HPD versus WD rats, there was enrichment of 114 operational taxonomic units (OTUs) and depletion of 188 OTUs. Of those, Akkermansia muciniphila (enriched on a HPD), an unclassified Clostridiales, a member of the RF39 order, and a Phascolarctobacterium were significantly associated with fat mass. The WD increased cytokine expression in the hypothalamus and dorsal medulla that was unchanged by switching to HPD. These data indicate that HPD reduces body fat and restores glucose homeostasis and CCK sensitivity, while not modifying brain inflammation. In addition, expansion of cecal Akkermansia muciniphila correlated to fat mass loss may represent a potential peripheral mechanism of HPD beneficial effects.

  11. Cholecystokinin actions in the parabrachial nucleus: effects on thirst and salt appetite

    NASA Technical Reports Server (NTRS)

    Menani, J. V.; Johnson, A. K.

    1998-01-01

    The present study investigated the effects of bilateral injections of the nonselective CCK receptor antagonist proglumide or CCK-8 into the lateral parabrachial nuclei (LPBN) on the ingestion of 0.3 M NaCl and water induced by intracerebroventricular injection of ANG II or by a combined treatment with subcutaneous furosemide (Furo) + captopril (Cap). Compared with the injection of saline (vehicle), bilateral LPBN injections of proglumide (50 micrograms . 200 nl-1 . site-1) increased the intake of 0.3 M NaCl induced by intracerebroventricular ANG II (50 ng/1 microliter). Bilateral injections of proglumide into the LPBN also increased ANG II-induced water intake when NaCl was simultaneously available, but not when only water was present. Similarly, the ingestion of 0.3 M NaCl and water induced by the treatment with Furo (10 mg/kg) + Cap (5 mg/kg) was increased by bilateral LPBN proglumide pretreatment. Bilateral CCK-8 (0.5 microgram . 200 nl-1 . site-1) injections into the LPBN did not change Furo + Cap-induced 0.3 M NaCl intake but reduced water consumption. When only water was available after intracerebroventricular ANG II, bilateral LPBN injections of proglumide or CCK-8 had no effect or significantly reduced water intake compared with LPBN vehicle-treated rats. Taken together, these results suggest that CCK actions in the LPBN play a modulatory role on the control of NaCl and water intake induced by experimental treatments that induce hypovolemia and/or hypotension or that mimic those states.

  12. Honokiol induces cell cycle arrest and apoptosis via p53 activation in H4 human neuroglioma cells.

    PubMed

    Guo, Yun-Bao; Bao, Xin-Jie; Xu, Song-Bai; Zhang, Xing-Dong; Liu, Hai-Yan

    2015-01-01

    To investigate the signal pathway of honokiol-induced apoptosis in H4 human neuroglioma cells and to evaluate whether p53 signaling and cell cycle arrest were involved in honokiol-treated H4 human neuroglioma cells. The cell viability was detected by the CCK8 assay. The cell apoptosis was assessed by annexin V-PI double-labeling staining and hoechst 33342 staining. The protein expression of cell cycle regulators and tumor suppressors were analyzed by western blotting. Treatment of H4 human neuroglioma cells with honokiol induced cell death in a dose-and time-dependent manner by using CCK8 assay. Consistent with the CCK8 assay, the flow cytometry results showed that the proportion of the apoptosis cells increased after honokiol when compared with untreated group. Moreover, H4 human neuroglioma cells exposed to honokiol, resulted in an accumulation of cells in S and G2/M phase. Apoptotic bodies were clearly observed in human neuroglioma cells when treated with honokiol and then stained with Hoechst 33342. The expression of Cyclin B1, CDC2 and cdc25C were downregulated, however, the expression of p-CDC2 and p-cdc25c was significantly upregulated when the neuroglioma cells were exposed to honokiol. Moreover, p53, p21 and Bax/Bcl-2 were significantly upregulated by honokiol treatment. These results confirmed that honokiol could induce apoptosis in human neuroglioma cells, the underlying molecular mechanisms, at least partially, through activation p53 signaling and induction of cell cycle arrest.

  13. Cytotoxicity evaluation of the whole protein extract from Bar-transgenic rice on Mus musculus lymphocytes.

    PubMed

    Lu, X B

    2017-01-23

    With the expanding demand for genetically modified (GM) rice, its safety evaluation is of great significance. Therefore, this study was carried out to assess the acute cytotoxicity of the whole protein extract from GM rice Bar68-1 in Mus musculus lymphocytes in vitro. Cell viability was determined by Cell Counting Kit-8 (CCK-8) and Neutral Red Uptake (NRU) tests. CCK-8 tests was carried out according to the manufacturer's instructions. Cell dehydrogenase (catalytic redox enzymes) activity was spectrophotometrically determined at 450 nm. The tests result were recorded immediately. NRU tests were completed under yellow light in a dark room according to an improved protocol. Lysosomal uptake of neutral red was spectrophotometrically determined at 540 nm and the results were recorded immediately. The results showed that the survival rate of M. musculus lymphocytes in the positive control group was significantly less than in the blank control group (P < 0.05). Moreover, an exposure- time-effect relationship was observed in the positive control group with CCK-8 and NRU tests. There was no significant difference in survival rate between GM rice Bar68-1group and non-GM rice D68 group (P > 0.05). The GM rice Bar68-1 group also did not show a higher survival rate than non-GM rice D68 group (P > 0.05). These results suggested that the whole protein extract from Bar68-1 and D68 were equivalent in their cytotoxicity, and GM rice Bar68-1 had no acute cytotoxic effect on M. musculus lymphocytes in vitro.

  14. Lack of ghrelin secretion in response to fasting in cholecystokinin-A (-1), -B (-2) receptor-deficient mice.

    PubMed

    Sakurai, Chihiro; Ohta, Minoru; Kanai, Setsuko; Uematsu, Hiroshi; Funakoshi, Akihiro; Miyasaka, Kyoko

    2006-12-01

    Cholecystokinin receptors (CCK-Rs) have been classified into two subtypes: CCK-AR (1R) and -BR (2R). We generated CCK-AR(-/-), CCK-BR(-/-), and CCK-AR(-/-)BR(-/-) mice and found that the gastric emptying of a liquid meal was increased in CCK-BR(-/-) and AR(-/-)BR(-/-) mice, compared with wild-type and CCK-AR(-/-) mice. Given that enhanced gastric emptying leads to eating, food intake after overnight fasting was examined, as was the effect of CCK-8S on food intake. Male mice 6-8 months of age were deprived of food for 16 h with free access to water, after which they were injected intraperitoneally (0.1 ml/mouse) with either vehicle or CCK-8 (0.3, 1.0, or 3.0 nmol/mouse), and their food intake was monitored for 4 h. CCK-8S inhibited food intake in wild-type and CCK-BR(-/-) mice, but not in CCK-AR(-/-) or AR(-/-)BR(-/-) mice. Unexpectedly, we observed a lower food intake in CCK-AR(-/-)BR (-/-) mice treated with vehicle than in mice of the other genotypes. To examine the mechanism of decrease in food intake in CCK-AR(-/-)BR(-/-) mice, the involvement of ghrelin was determined in wild-type and CCK-AR(-/-)BR(-/-) mice. Fasting plasma ghrelin levels were significantly lower in CCK-AR (-/-)BR(-/-) mice than in wild-type mice, and no increase in response to fasting was observed in CCK-AR(-/-)BR(-/-) mice. An administration of acyl-ghrelin produced a small increase in food intake in CCK-AR(-/-)BR(-/-) mice, but not to the levels of wild-type mice. In conclusion, CCK-AR(-/-)BR(-/-) mice showed lower food intake as well as lower response to exogenous ghrelin, and a lower plasma ghrelin level after fasting, though which receptor is more important is unknown.

  15. Evaluation of myosin light chain phosphorylation in isolated pancreatic acini

    SciTech Connect

    Burnham, D.B.; Soeling, H.D.; Williams, J.A. Universitaet Goettingen )

    1988-01-01

    The role of contractile proteins in secretory granule exocytosis was evaluated by determining whether myosin light chain phosphorylation was altered during stimulation of secretion in mouse pancreatic acini. Acinar myosin was purified by extraction into isosmotic sucrose solution containing 40 mM pyrophosphate followed by ammonium sulfate precipitation and Sepharose 4B-CL chromatography. Myosin was eluted as a single peak of K{sup +}-EDTA ATPase activity and was purified over 2,000-fold to a final ATPase specific activity of 0.96 {mu}mol{center dot}min{sup {minus}1}{center dot}mg protein {sup {minus}1}. Three major myosin subunits of apparent M{sub r} of 200,000, 20,000, and 17,000 were present in the purified myosin preparation. A fourth protein of M{sub r} 21,000 was also present. Purification of myosin from {sup 32}P-labeled acini revealed that M{sub r} 200,000, 21,000, and 20,000 proteins to be heavily labeled. The effect of cholecystokinin octapeptide (CCK-8) on myosin phosphorylation was studied after isolation of myosin from {sup 32}P-labeled acinar lysates by immunoprecipitation. Treatment of acini for 1-10 min with a concentration of CCK-8 that gives a maximal secretory response caused a 25-40% increase in light chain labeling. Treatment with a supramaximal CCK-8 concentration produced a 50-80% increase in light chain labeling. Phosphorylation of myosin heavy chain was not significantly affected by secretagogue treatment. These results indicate that stimulation of pancreatic acinar secretion is accompanied by an increase in myosin light chain phosphorylation.

  16. Cholecystokinin actions in the parabrachial nucleus: effects on thirst and salt appetite

    NASA Technical Reports Server (NTRS)

    Menani, J. V.; Johnson, A. K.

    1998-01-01

    The present study investigated the effects of bilateral injections of the nonselective CCK receptor antagonist proglumide or CCK-8 into the lateral parabrachial nuclei (LPBN) on the ingestion of 0.3 M NaCl and water induced by intracerebroventricular injection of ANG II or by a combined treatment with subcutaneous furosemide (Furo) + captopril (Cap). Compared with the injection of saline (vehicle), bilateral LPBN injections of proglumide (50 micrograms . 200 nl-1 . site-1) increased the intake of 0.3 M NaCl induced by intracerebroventricular ANG II (50 ng/1 microliter). Bilateral injections of proglumide into the LPBN also increased ANG II-induced water intake when NaCl was simultaneously available, but not when only water was present. Similarly, the ingestion of 0.3 M NaCl and water induced by the treatment with Furo (10 mg/kg) + Cap (5 mg/kg) was increased by bilateral LPBN proglumide pretreatment. Bilateral CCK-8 (0.5 microgram . 200 nl-1 . site-1) injections into the LPBN did not change Furo + Cap-induced 0.3 M NaCl intake but reduced water consumption. When only water was available after intracerebroventricular ANG II, bilateral LPBN injections of proglumide or CCK-8 had no effect or significantly reduced water intake compared with LPBN vehicle-treated rats. Taken together, these results suggest that CCK actions in the LPBN play a modulatory role on the control of NaCl and water intake induced by experimental treatments that induce hypovolemia and/or hypotension or that mimic those states.

  17. PKC-mediated inhibitory feedback of the cholecystokinin 1 receptor controls the shape of oscillatory Ca²⁺ signals.

    PubMed

    Willems, Peter H G M; Pahle, Jürgen; Stalpers, Xenia L; Mugahid, Douaa; Nikolaew, Alexander; Koopman, Werner J H; Kummer, Ursula

    2015-06-01

    Translation of extracellular hormonal input into cellular responses is often mediated by repetitive increases in cytosolic free Ca(2+) concentration ([Ca(2+) ]c ). Amplitude, duration and frequency of these so-called [Ca(2+) ]c oscillations then carry information about the nature and concentration of the extracellular signalling molecule. At present, there are different hypotheses concerning the induction and control of these oscillations. Here, we investigated the role of agonist-induced receptor phosphorylation in this process using Chinese hamster ovary cells stably expressing a variant of the cholecystokinin 1 receptor (CCK1R) lacking the four consensus sites for protein kinase C (PKC) phosphorylation and deficient in CCK-induced receptor phosphorylation (CCK1R-mt cells). In the presence of cholecystokinin-(26-33)-peptide amide (CCK-8), these cells displayed Ca(2+) oscillations with a much more pronounced bursting dynamics rather than the dominant spiking dynamics observed in Chinese hamster ovary cells stably expressing the wild-type CCK1R. The bursting behaviour returned to predominantly spiking behaviour following removal of extracellular Ca(2+) , suggesting that CCK-8-induced, PKC-mediated CCK1R phosphorylation inhibits Ca(2+) influx across the plasma membrane. To gain mechanistic insight into the underlying mechanism we developed a mathematical model able to reproduce the experimental observations. From the model we conclude that binding of CCK-8 to the CCK1R leads to activation of PKC which subsequently phosphorylates the receptor to inhibit the receptor-mediated influx of Ca(2+) across the plasma membrane. Receptor-specific differences in this feedback mechanism may, at least in part, explain the observation that different agonists evoke [Ca(2+) ]c oscillations with different kinetics in the same cell type. © 2015 FEBS.

  18. Synthesis of cyclic analogues of cholecystokinin highly selective for central receptors.

    PubMed

    Rodriguez, M; Amblard, M; Galas, M C; Lignon, M F; Aumelas, A; Martinez, J

    1990-05-01

    Cyclic CCK analogues in which positions 28 and 31 have been replaced by lysine residues and whose side chains are bridged by a succinic moiety, were synthesized. They were tested for their ability to inhibit the binding of 125I-BH-CCK-8 to isolated rat pancreatic acini and to guinea pig brain membranes. These cyclic CCK-analogues were compared to the potent CCK analogue Boc-[Nle28,31]-CCK-7 and to Boc-Trp-Leu-Asp-Phe-NH2, analogue of CCK-4. These cyclic compounds appeared to be highly selective for central CCK receptors.

  19. Synthesis and biological activity of some partially modified retro-inverso analogues of cholecystokinin.

    PubMed

    Rodriguez, M; Galas, M C; Lignon, M F; Mendre, C; Laur, J; Aumelas, A; Martinez, J

    1989-10-01

    Syntheses of some partially modified retro-inverso analogues of the C-terminal octa- or heptapeptide of cholecystokinin are described. These analogues (in which the C-terminal carboxamide was deleted or not) were obtained by reverting one or several peptide bonds in the parent molecule. All these compounds were able to inhibit binding of labeled CCK-8 to rat pancreatic acini and guinea pig brain membranes and to stimulate amylase release from rat pancreatic acini with various potencies. Some of these derivatives reproduce only part of the biological response of CCK on amylase release.

  20. 2-Phenylethyl ester and 2-phenylethyl amide derivative analogues of the C-terminal hepta- and octapeptide of cholecystokinin.

    PubMed

    Fulcrand, P; Rodriguez, M; Galas, M C; Lignon, M F; Laur, J; Aumelas, A; Martinez, J

    1988-11-01

    Syntheses of analogues of the C-terminal octa- and heptapeptide of cholecystokinin are described. These analogues were obtained by replacing the C-terminal phenylalanine residue by 2-phenylethyl alcohol or by 2-phenylethylamine derivatives and by replacing the tryptophan residue by a D-tryptophan. The CCK-derivatives were tested for their ability to inhibit binding of labeled CCK-8 to rat pancreatic acini and to guinea pig brain membranes, and for their action on stimulation of amylase release from rat pancreatic acini. Some of these derivatives appeared to exhibit only part of the CCK-activity on amylase release, the D-Trp analogues behaving as CCK-antagonists.

  1. Electrophysiological evidence for distinct vagal pathways mediating CCK-evoked motor effects in the proximal versus distal stomach

    PubMed Central

    Okano-Matsumoto, Shiho; McRoberts, James A; Taché, Yvette; Adelson, David W

    2011-01-01

    Intravenous cholecystokinin octapeptide (CCK-8) elicits vago-vagal reflexes that inhibit phasic gastric contractions and reduce gastric tone in urethane-anaesthetized rats. A discrete proximal subdivision of the ventral gastric vagus nerve (pVGV) innervates the proximal stomach, but the fibre populations within it have not been characterized previously. We hypothesized that i.v. CCK-8 injection would excite inhibitory efferent outflow in the pVGV, in contrast to its inhibitory effect on excitatory efferent outflow in the distal subdivision (dVGV), which supplies the distal stomach. In each VGV subdivision, a dual-recording technique was used to record afferent and efferent activity simultaneously, while also monitoring intragastric pressure (IGP). CCK-8 dose dependently (100–1000 pmol kg−1, i.v.) reduced gastric tone, gastric contractile activity and multi-unit dVGV efferent discharge, but increased pVGV efferent firing. Single-unit analysis revealed a minority of efferent fibres in each branch whose response differed in direction from the bulk response. Unexpectedly, efferent excitation in the pVGV was significantly shorter lived and had a significantly shorter decay half-time than did efferent inhibition in the dVGV, indicating that distinct pathways drive CCK-evoked outflow to the proximal vs. the distal stomach. Efferent inhibition in the dVGV began several seconds before, and persisted significantly longer than, simultaneously recorded dVGV afferent excitation. Thus, dVGV afferent excitation could not account for the pattern of dVGV efferent inhibition. However, the time course of dVGV afferent excitation paralleled that of pVGV efferent excitation. Similarly, the duration of CCK-8-evoked afferent responses recorded in the accessory celiac branch of the vagus (ACV) matched the duration of dVGV efferent responses. The observed temporal relationships suggest that postprandial effects on gastric complicance of CCK released from intestinal endocrine cells may

  2. Pharmacological evaluation of IQM-95,333, a highly selective CCKA receptor antagonist with anxiolytic-like activity in animal models

    PubMed Central

    Ballaz, Santiago; Barber, Ana; Fortuño, Ana; Del Río, Joaquín; Martín-Martínez, Mercedes; Gómez-Monterrey, Isabel; Herranz, Rosario; González-Muñiz, Rosario; García-López, Maria-Teresa

    1997-01-01

    The pyridopyrimidine derivative IQM-95,333 ((4aS,5R)-2-benzyl-5-[Nα-tert-butoxicarbonyl)L-tryptophyl]amino-1,3dioxoperhydropyrido[1,2-c]pyrimidine), a new non-peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluated in vitro and in vivo in comparison with typical CCKA and CCKB receptor antagonists, such as devazepide, lorglumide, L-365,260 and PD-135,158. IQM-95,333 displaced [3H]-CCK-8S binding to CCKA receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKB receptors was negligible (IC50>10 μM). IQM-95,333 was a more selective CCKA receptor ligand than devazepide and other CCKA receptor antagonists. Like devazepide, IQM-95,333 was a more potent antagonist of CCK-8S- than of CCK-4-induced contraction of the longitudinal muscle from guinea-pig ileum, suggesting selective antagonism at CCKA receptors. IQM-95,333 and devazepide were also potent inhibitors of CCK-8S-stimulated amylase release from isolated pancreatic acini, a CCKA receptor-mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates. Low doses (50–100 μg kg−1, i.p.) of IQM-95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK-8S, two effects associated with stimulation of peripheral CCKA receptors. IQM-95,333 showed an anxiolytic-like profile in the light/dark exploration test in mice over a wide dose range (10–5,000 μg kg−1). Typical CCKA and CCKB antagonists, devazepide and L-365,260 respectively, were only effective within a more limited dose range. In a classical conflict paradigm for the study of anxiolytic drugs, the punished-drinking test, IQM-95,333, devazepide and L-365,260 were effective within a narrow dose range. The dose-response curve for the three drugs was biphasic, suggesting that

  3. Taste matters - effects of bypassing oral stimulation on hormone and appetite responses.

    PubMed

    Spetter, Maartje S; Mars, Monica; Viergever, Max A; de Graaf, Cees; Smeets, Paul A M

    2014-10-01

    The interaction between oral and gastric signals is an important part of food intake regulation. Previous studies suggest that bypassing oral stimulation diminishes the suppression of hunger and increases gastric emptying rate. However, the role of appetite hormones, like cholecystokinin-8 and ghrelin, in this process is still unclear. Our objective was to determine the contributions of gastric and oral stimulation to subsequent appetite and hormone responses and their effect on ad libitum intake. Fourteen healthy male subjects (age 24.6±3.8y, BMI 22.3±1.6kg/m(2)) completed a randomized, single-blinded, cross-over experiment with 3 treatment-sessions: 1) Stomach distention: naso-gastric infusion of 500mL/0kJ water, 2) Stomach distention with caloric content: naso-gastric infusion of 500mL/1770kJ chocolate milk, and 3) Stomach distention with caloric content and oral exposure: oral administration of 500mL/1770kJ chocolate milk. Changes in appetite ratings and plasma glucose, insulin, cholecystokinin-8, and active and total ghrelin concentrations were measured at fixed time-points up to 30min after infusion or oral administration. Subsequently, subjects consumed an ad libitum buffet meal. Oral administration reduced appetite ratings more than both naso-gastric infusions (P<0.0001). Gastric infusion of a caloric load increased insulin and cholecystokinin-8 and decreased total ghrelin concentrations more than ingestion (all P<0.0001). No differences in active ghrelin response were observed between conditions. Ad libitum intake did not differ between oral and gastric administration of chocolate milk (P>0.05). Thus, gastric infusion of nutrients induces greater appetite hormone responses than ingestion does. These data provide novel and additional evidence that bypassing oral stimulation not only affects the appetite profile but also increases anorexigenic hormone responses, probably driven in part by faster gastric emptying. This confirms the idea that learned

  4. Immunohistochemical study on the neuroendocrine system of the digestive tract of turbot, Scophthalmus maximus (L.), infected by Enteromyxum scophthalmi (Myxozoa).

    PubMed

    Bermúdez, R; Vigliano, F; Quiroga, M I; Nieto, J M; Bosi, G; Domeneghini, C

    2007-03-01

    In recent years a new parasite, causing severe losses, has been detected in farmed turbot, Scophthalmus maximus (L.), in Northwestern Spain. Dead fish showed emaciation and cachexia caused by severe necrotizing enteritis, which affected all areas of the digestive tract. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. This study was designed to assess the response of the turbot neuroendocrine system against E. scophthalmi infection. Immunohistochemical tests were applied to sections of the gastrointestinal tract of uninfected and E. scophthalmi-infected turbot, and the presence of cholecystokinin (CCK-8), serotonin (5-HT), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) were documented. A higher abundance of both endocrine epithelial cells (ECs) and nerve cell bodies and fibres for CCK-8, 5-HT and SP were recorded in the gastrointestinal tract of infected turbot, whereas VIP-like substance decreased. The results indicate that E. scophthalmi infection in turbot induced changes in the neuroendocrine system, which may cause alterations in gut motility, electrolyte and fluid secretion, and vascular and immune functions.

  5. Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

    NASA Astrophysics Data System (ADS)

    Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

    2016-01-01

    To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation.

  6. Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

    SciTech Connect

    Matozaki, T.; Sakamoto, C.; Nagao, M.; Nishisaki, H.; Konda, Y.; Nakano, O.; Matsuda, K.; Wada, K.; Suzuki, T.; Kasuga, M. )

    1991-02-14

    The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of {sup 125}I-CCK-octapeptide (CCK8). At 30{degrees}C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.

  7. Protective effect of bone marrow derived mesenchymal stem cells in lipopolysaccharide-induced acute lung injury mediated by claudin-4 in a rat model

    PubMed Central

    Zheng, Yueliang; Cai, Wenwei; Zhou, Shengang; Xu, Liming; Jiang, Chengxing

    2016-01-01

    Our study aims to investigate the effects of bone marrow derived mesenchymal stem cells (BM-MSCs) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) as well as the underlying mechanism. In our study, Wistar rats were randomly divided into four groups: control group; ALI group; ALI+MSCs group and ALI+MSCs claudin-4 siRNA group. MRC-5 and BEAS-2B cell lines were used for in vitro assay. Flow cytometry, western blot, hematoxylin and eosin (H&E) staining, CCK-8 assay, enzyme-linked immunosorbent assay (ELISA) were involved to measure the pathological changes in lung tissues. Results showed that in vivo MSCs administration significantly attenuated pulmonary edema (wet/dry ratio), inflammation cytokines levels (TGF-α), pathological alternations and cell apoptosis which were mediated by claudin-4 in LPS-induced acute lung injury in rats. In vitro experiment showed that hypoxia could induce the expression of claudin-4 in MSCs, and MSCs treatment showed significantly enhanced cell viability (by CCK-8 assay) and reduced cell apoptosis. In conclusion, the present study demonstrated that BM-MSCs can protect against LPS-induced ALI in vivo and in vitro, at least partly mediated by claudin-4. PMID:27725857

  8. Antiproliferative effects of 5-fluorouracil and oxaliplatin in colon cancer cell lines: comparison of three different cytotoxicity assays.

    PubMed

    Failli, A; Legitimo, A; Orsini, G; Castagna, M; Spisni, R; Miccoli, P; Consolini, R

    2013-01-01

    Adjuvant therapy in colorectal cancer has evolved to become the standard of care, whereas the tumor capability of activating effective mechanisms of defence against both chemical and physical cytotoxic agents represents a serious obstacle to the successful therapy of human tumors. Therefore, the possibility to have an assay useful to measure the drug sensitivity of tumor cells has a great importance. A number of cytotoxicity assays are currently available, each of them using a specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. The purpose of this study is to compare, under identical experimental conditions, three common cytotoxicity assays (ATP-lite, MTT and CCK-8 assays) in the assessment of the anti-proliferative effects of 5-fluorouracil (5-FU) and oxaliplatin (OHP) on three colon cancer cell lines (WiDr, SW620 and HT-29). Regarding 5-FU, the three assays were found to be significantly correlated with a moderate or high correlation coefficient, whereas in the case of OHP we found different outcomes among the assays. Our study demonstrates that the CCK-8 is the most sensitive assay for detecting changes of cell viability, suggesting that the viability measured in cells after drug exposure depends on several parameters like the drug used, the biological characteristics of the target cell and the specific approach employed by the method to detect distinct cell growth and metabolic functions.

  9. Notch1/3 and p53/p21 are a potential therapeutic target for APS-induced apoptosis in non-small cell lung carcinoma cell lines.

    PubMed

    Zhang, Jing-Xi; Han, Yi-Ping; Bai, Chong; Li, Qiang

    2015-01-01

    Previous studies have shown that Astragalus polysaccharide (APS) can be applied to anti-cancer. However, the mechanism by which APS mediate this effect is unclear. In the present study, APS-mediated NSCLC cell apoptosis was investigated through the regulation of the notch signaling pathway. The cell viability was detected by the CCK8 assay. The mRNA and protein expression of notch1/3 and tumor suppressors were analyzed by RT-PCR and western blotting, respectively. The mRNA and protein of notch1 and notch3 were significantly up-regulated in tumor tissues as compared to non-tumor adjacent tissues. Treatment of human NSCLC cells with APS induced cell death in a dose-and time-dependent manner by using CCK8 assay. The mRNA and protein expression of notch1 and notch3 were significantly lower in NSCLC cells with APS treatment than that in control group. Moreover, western blotting analysis showed that treatment of H460 cells with APS significantly increased the pro-apoptotic Bax and caspase 8 levels, decreased the anti-apoptotic Bcl-2 level. Furthermore, p53, p21 and p16 were obviously up-regulated by APS treatment in H460 cell. This study demonstrated that APS-treated could inhibit proliferation and promote cell apoptosis, at least partially, through suppressing the expression of notch1 and notch3 and up-regulating the expression of tumor suppressors in H460 NSCLC cell lines.

  10. Inhibitory effects of ethanol on phosphatidylinositol breakdown in pancreatic acini

    SciTech Connect

    Towner, S.J.; Peppin, J.F.; Tsukamoto, H.

    1986-03-01

    Recently the physiological relationship between the phospholipid effect and secretagogue-induced cellular function has begun to be understood. In this study, the authors investigated acute and chronic effects of ethanol on phosphatidylinositol (PI) synthesis and breakdown in pancreatic acini. Five pairs of male Wistar rats were intragastrically infused for 30 days with high fat diet (25% total calories) plus ethanol or isocaloric dextrose. After intoxication, isolated in HEPES media, followed by 30 min incubation with CCK-8 (0, 100, 300 or 600 pM) and ethanol (0 or 100 mM). Acinar lipids were extracted and counted for labeled PI. Incorporation of /sup 3/H-inositol into alcoholic acinar PI was reduced to 38.2% of that in controls. A percent maximal PI break down by CCK-8 was similar in the two groups (13-24% of basal). However, the magnitude of PI breakdown was markedly lower in alcoholic acini (482 vs 1081 dpm) due to the decreased PI synthesis rate. The presence of 100 mM ethanol in the media further inhibited the breakdown by 50% in this group. These results strongly indicate that chronic ethanol intoxication inhibits PI synthesis and breakdown in pancreatic acini, and that this inhibition can be potentiated by acute ethanol administration.

  11. Preparation and preliminary in vitro evaluation of a bFGF-releasing heparin-conjugated poly(ε-caprolactone) membrane for guided bone regeneration.

    PubMed

    Cao, Cong; Song, Ying; Yao, Qianqian; Yao, Yang; Wang, Tianlu; Huang, Bo; Gong, Ping

    2015-01-01

    In an effort to improve guided bone regeneration (GBR), we successfully fabricated a novel basic fibroblast growth factor (bFGF)-releasing heparin-conjugated poly(ε-caprolactone) membrane (hep-PCL/bFGF). This material has a porous microstructure with smooth and rough pore walls before and after heparinization, respectively. Our FTIR analyses indicated that chemical bonds were formed between PCL and heparin with a new amide C=O band at 1660 cm(-1) and a band at 3400 cm(-1) that can be attributed to -OH stretching in cross-linked heparin. We showed that bFGF was released from hep-PCL/bFGF in a continuous pattern, which remained for 3 weeks. We evaluated MG63 cell proliferation and biocompatibility of GBR membrane by a CCK-8 assay and a live/dead assay. The CCK-8 results revealed that the hep-PCL/bFGF group had superiority compared to other groups. Furthermore, cell morphology of hep-PCL membrane exhibited larger projected areas than those of PCL surfaces based on scanning electron microscopy analysis and immunofluorescent staining of cell cytoskeleton and vinculin expression. Our alkaline phosphatase activity assay also confirmed better performance of the hep-PCL/bFGF group. These results suggested that this novel hep-PCL/bFGF membrane is suitable for osteoblast-like cells to attach, proliferate, and differentiate. Therefore, the hep-PCL/bFGF membrane has potential to be a biodegradable membrane for GBR and warrants further investigation.

  12. [Astaxanthin inhibits proliferation and promotes apoptosis of A549 lung cancer cells via blocking JAK1/STAT3 pathway].

    PubMed

    Wu, Chuntao; Zhang, Jinji; Liu, Tienan; Jiao, Guimei; Li, Changzai; Hu, Baoshan

    2016-06-01

    Objective To investigate the anti-tumor effects of astaxanthin on A549 lung cancer cells and the related mechanisms. Methods A549 cells were cultured with various concentrations of astaxanthin (20, 40, 60, 80, 100 μmol/L), and DMSO at the same concentrations served as vehicle controls. The viability of A549 cells was detected by CCK-8 assay; cell cycle and apoptosis were observed by flow cytometry; and the expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), signal transducers and activators of transcription 3 (STAT3), and Janus kinase 1 (JAK1) were evaluated by Western blotting. Results CCK-8 assay showed that astaxanthin decreased the proliferation of A549 cells in a dose-dependent manner. Flow cytometry showed that astaxanthin increased the number of cells in the G0/G1 phase and induced apoptosis in A549 cells. Western blotting showed that astaxanthin up-regulated the expression of Bax and down-regulated the expressions of Bcl-2, STAT3 and JAK1. Conclusion Astaxanthin functions as a potent inhibitor of A549 lung cancer cell growth by targeting JAK1/STAT3 signaling pathway.

  13. [Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells].

    PubMed

    Li, Ji; Liu, Zuojin

    2015-12-01

    To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0×10(8) TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells. Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (P<0.05). In Kupffer cells, lentivirus-mediated transfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.

  14. [Effect of overexpression of CAV1 mediated by lentivirus on proliferation and apoptosis of HL-60 cells].

    PubMed

    Ma, Wei; Wang, Di-Di; Wang, Zhao; Zhu, Gui-Ming; Zhang, Peng-Xia

    2013-08-01

    This study was purposed to explore the effect of lentivirus-mediated CAV1 overexpression on proliferation and apoptosis in HL-60 cells. Recombinant lentiviral expression vector pcDNA-EF1-CAV1 was constructed, and cotransfected the 293TN cells with a mixture of pPACK packaging plasmids. Then collecting virus suspension infects the HL-60 cells, which make CAV1 gene stable transfection and high expression in the cells. The CAV1 protein expression status in HL-60 cells transfected was evaluated through Western blot method. Proliferative activity and apoptosis of HL-60 cells before and after transfection were detected by CCK-8 method and flow cytometry, respectively. The results showed that the PCR-positive clone screening and results of nucleotide sequencing confirmed that the CAV1 gene inserted into the expression vector pcDNA-EF1-GFP correctly, recombinant lentiviral particles Lv-CAV1 transfected HL-60 cells successfully and with transfection rate up to 90%. The result of Western blot showed that CAV1 protein expression in HL-60 cells significantly increased at 48 hours after transfection. CCK-8 result indicated that cell proliferation activity increased at 48 h after transfection (P < 0.05), flow cytometry testing results displayed that apoptosis rate of HL-60 cells obviously decreased after transfection (P < 0.01). It is concluded that the overexpression of CAV1 in HL-60 cells can inhibit cell proliferation activity and promote cell apoptosis.

  15. Studying the impact of S9 on cyto-genotoxicity of cigarette smoke in human peripheral blood lymphocytes in vitro.

    PubMed

    Jianlin, Lou; Guohai, Chu; Guojun, Zhou; Jian, Jiang; Fangfang, Huang; Juanjuan, Xu; Shu, Zheng; Zhijian, Chen; Wei, Jiang; Yezhen, Lu; Xiaoxue, Li; Jiliang, He

    2009-09-01

    In present study, human lymphocytes were exposed to cigarette smoke condensates (CSCs) at the doses of 25, 50, 75, 100 and 125 μg ml(-1) with and without S9, and the cyto-genotoxic effects were detected with CCK-8, cell apoptosis and micronucleus assays. DNA repair kinetics was observed with comet assay. Our results indicated that the cell viability decreased with CSCs doses, the percentages of apoptosis cell and the frequencies of micronuclei increased with CSCs doses, and DNA damage of human lymphocytes induced by CSCs could be basically repaired within 240 min. However, the cytotoxicity induced by CSCs +S9 was significantly lower than that induced by CSCs -S9 in CCK-8 and cell apoptosis assays, and the DNA repair speed in +S9 group was quicker than that in -S9 group. In conclusion, S9 may affect not only the cyto-genotoxicity of CSCs but also the repair process of DNA damage induced by CSCs in lymphocytes.

  16. Promotion of Astragaloside IV for EA-hy926 Cell Proliferation and Angiogenic Activity via ERK1/2 Pathway.

    PubMed

    Wang, Shi; Chen, Jiandong; Fu, Yuxuan; Chen, Xiaohu

    2015-06-01

    The aim of this study was to determine the pro-angiogenic effects of Astragaloside IV (AS-IV) in vitro and reveal the potential mechanisms. A kind of human umbilical vein endothelial cells (HUVECs), named EA-hy926 cells, were treated with various dosages of AS-IV. We then utilized Cell Counting Kit-8 (CCK-8), real-time PCR and Western blot to detect EA-hy926 cells' proliferation and proangiogenic effect from AS-IV. Data showed that AS-IV promoted EA-hy926 cells proliferation, as assessed by CCK-8. The AS-IV was also associated with an increased tube formation and upregulation of vascular endothelial growth factor (VEGF) mRNA and protein in a dose-dependent manner. Interestingly, the influence of AS-IV on cell proliferation and angiogenisis could be abolished by inhibitor PD98059 through suppressed extracellular signal regulated protein kinases1/2 (ERK1/2) phosphorylation. These data demonstrated that the AS-IV activated the ERK1/2 pathway to control VEGF synthesis. Our findings conclude that the AS-IV promotes EA-hy926 cells proliferation and angiogenesis through ERK1/2 pathway, and it is also a regulator of VEGF.

  17. Effects of TGF-β1 on the Proliferation and Apoptosis of Human Cervical Cancer Hela Cells In Vitro.

    PubMed

    Tao, Ming-Zhu; Gao, Xia; Zhou, Tie-Jun; Guo, Qing-Xi; Zhang, Qiang; Yang, Cheng-Wan

    2015-12-01

    To investigate the effects of TGF-β1 on the proliferation and apoptosis of cervical cancer Hela cells in vitro. Human cervical cancer Hela cells were cultured in vitro and divided into the experimental and control groups. In the experimental groups, Hela cells were stimulated with different concentrations of TGF-β1 (0.01, 0.1, 1, and 10 ng/mL), while Hela cells cultured in serum-free medium without TGF-β1 were used as controls. The CCK8 method was adopted to detect the effect of TGF-β1 on Hela cell proliferation, and flow cytometry was used to determine cell apoptosis 72 h after TGF-β1 treatment. Compared with the control group, the CCK-8 tests showed that different concentrations of TGF-β1 had no obvious effect on Hela cell proliferation 24 h after treatment (P > 0.05). However, upon 48 or 72 h of treatment, TGF-β1 significantly inhibited the proliferation of Hela cells in a time- and dose-dependent manner (P < 0.05). The flow cytometry results indicated that TGF-β1 influenced the apoptosis of human cervical cancer Hela cells in a dose-dependent manner after 72 h of treatment (P < 0.05). TGF-β1 significantly inhibited the growth and induced the apoptosis of human cervical Hela cells in vitro.

  18. Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway.

    PubMed

    Li, Xiaolei; Sun, Yu; Chen, Hui; Zhu, Gengyao; Liang, Yuan; Wang, Qiang; Wang, Jingcheng; Yan, Lianqi

    2016-06-15

    Hydroxycamptothecin (HCPT) has been proven to prevent intraarticular scar adhesion, but the mechanism is still unclear. ER stress is known to participate in many diseases, and the IRE-1 signal pathway has been reported in fibrotic diseases. The aim of this study was to illustrate the mechanism of HCPT-induced apoptosis in fibroblasts and the prevention of intraarticular scar adhesion. The effects of HCPT on fibroblasts were determined by CCK-8 assay, Hoechst staining and Western blot. The effect of HCPT on intraarticular scar adhesion was detected by macroscopic evaluation, hydroxyproline content, histological evaluation, fibroblast counting and immunohistochemical analysis. HCPT induced apoptosis of fibroblasts, according to CCK-8 assays, Hoechst staining and Western blot analysis. As the concentration of HCPT increased, the expressions of glucose-regulated protein 78 (GRP78), inositol-requiring kinase1 (IRE-1), C/EBP homologous protein (CHOP) and Bax were all increased, but the expression of Bcl-2 was decreased. Knockdown of IRE-1 alleviated the HCPT-induced apoptosis in our fibroblast model. HCPT could prevent intraarticular scar adhesion, according to the results of macroscopic evaluation, hydroxyproline content, histological evaluation and fibroblast counting in a rabbit model. Immunohistochemical analysis showed that IRE-1 expression increased as the concentration increased. The present study showed that the IRE-1 signal pathway might be involved in HCPT-induced apoptosis of fibroblast and might play a role in preventing intraarticular scar adhesion. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Novel porous graphene oxide and hydroxyapatite nanosheets-reinforced sodium alginate hybrid nanocomposites for medical applications

    SciTech Connect

    Xiong, Guangyao; Luo, Honglin; Zuo, Guifu; Ren, Kaijing; Wan, Yizao

    2015-09-15

    Graphene oxide (GO) and hydroxyapatite (HAp) are frequently used as reinforcements in polymers to improve mechanical and biological properties. In this work, novel porous hybrid nanocomposites consisting of GO, HAp, and sodium alginate (SA) have been prepared by facile solution mixing and freeze drying in an attempt to obtain a scaffold with desirable mechanical and biological properties. The as-prepared porous GO/HAp/SA hybrid nanocomposites were characterized by SEM, XRD, FTIR, TGA, and mechanical testing. In addition, preliminary cell behavior was assessed by CCK8 assay. It is found that the GO/HAp/SA nanocomposites show improved compressive strength and modulus over neat SA and HAp/SA nanocomposites. CCK8 results reveal that the GO/HAp/SA nanocomposites show enhanced cell proliferation over neat SA and GO/SA nanocomposite. It has been demonstrated that GO/HAp20/SA holds promise in bone tissue engineering. - Graphical abstract: Display Omitted - Highlights: • Graphene oxide (GO), hydroxyapatite (HAp), and alginate (SA) nanocomposites were fabricated. • The novel porous composites were prepared by solution mixture and freeze drying. • The GO/HAp/SA had porous structure with porosity > 85% and pore size > 150 μm. • The GO/HAp/SA exhibited improved mechanical properties over HAp/SA counterparts. • The GO/HAp/SA showed enhanced cell proliferation over GO/SA counterparts.

  20. CCK-5: sequence analysis of a small cholecystokinin from canine brain and intestine

    SciTech Connect

    Shively, J.; Reeve, J.R. Jr.; Eysselein, V.E.; Ben-Avram, C.; Vigna, S.R.; Walsh, J.H.

    1987-02-01

    The purpose of this study is to purify and to characterize chemically cholecystokinin (CCK)-like peptides present in brain and gut extracts that elute from gel filtration after the octapeptide. Canine small intestinal mucosa and brain were boiled in water and then extracted in cold trifluoroacetic acid, and cholecystokinin-like immunoreactivity was determined by carboxyl-terminal specific radioimmunoassay. Gel permeation chromatography on Sephadex G-50 revealed a form of CCK apparently smaller than CCK-8. Microsequence analysis showed that the amino terminal primary sequence of this small CCK was Gly-Trp-Met-Asp. Immunochemical and chromatographic analysis indicated that the carboxyl-terminal residue was Phe-NH2 and thus the full sequence is Gly-Trp-Met-Asp-Phe-NH2. An antibody that recognizes synthetic CCK-8, CCK-5, and CCK-equally did not reveal the presence of significant amounts of CCK-4. These results indicate that CCK-5 is the major CCK form smaller than the octapeptide present in brain and small intestine. This finding, coupled with the demonstration by others that CCK-5 interacts with high-affinity brain CCK receptors, indicates that CCK-5 may play a physiological role in brain function.

  1. [IL-37 inhibits the proliferation, invasion and migration of SMMC-7721 cells in vitro].

    PubMed

    Zhang, Guoqing; Tang, Chengyoug; Tan, Jingyu; Liu, Rui; Zhou, Mi; Wu, Zhongjun

    2015-10-01

    To analyze the impact of interleukin-37 (IL-37) on proliferation, migration and invasion of SMMC-7721 hepatocellular carcinoma cells and the related molecular mechanism. SMMC-7721 cells cultured in vitro were divided into the (50, 100, 200, 500) ng/mL recombinant human IL-37 (rhIL-37) treated groups and control group. Cell proliferation ability was detected by CCK-8 assay. Wound healing assay was used to observe cell migration ability. Transwell(TM) assay was performed to test the cell invasion ability. The levels of signal transducers and activators of transcription 3 (STAT3), phospho-STAT3 (p-STAT3) and matrix metalloproteinase-2 (MMP-2) were determined using Western blotting. CCK-8 assay showed that rhIL-37 inhibited the proliferation of SMMC-7721 cells. Wound healing assay revealed that rhIL-37 inhibited the migration of SMMC-7721 cells. Transwell(TM) indicated that rhIL-37 inhibited the invasion of SMMC-7721 cells. Western blotting demonstrated that rhIL-37 down-regulated the expressions of p-STAT3 and MMP-2. IL-37 can inhibit the proliferation, migration and invasion of SMMC-7721 cells. It may be related with the down-regulation of p-STAT3 and MMP-2.

  2. Effect of the lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) on the alpha-amylase secretion of rat pancreas in vitro and in vivo.

    PubMed

    Mikkat, U; Damm, I; Schröder, G; Schmidt, K; Wirth, C; Weber, H; Jonas, L

    1998-05-01

    Lectins are able to bind to cholecystokinin (CCK) receptors and other glycosylated membrane proteins. The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) are used for affinity chromatography to isolate the highly glycosylated CCK-A receptor of pancreatic acinar cells. According to the working hypothesis that lectin binding to the CCK receptor should alter the ligand-receptor interaction, the effect of WGA and UEA-I on CCK-8-induced enzyme secretion was studied on isolated rat pancreatic acini in vitro. In vitro both lectins showed a dosage-dependent inhibition of CCK-8-induced alpha-amylase secretion of acini over 60 min. WGA showed a strong inhibitory effect on amylase secretion, approximately 40%, in vitro. UEA-I caused a smaller, but significant decrease, approximately 20%, in enzyme secretion of isolated acini. Additionally, both lectins inhibited cerulein/secretin- or cerulein-induced pancreatic secretion of rats in vivo, but not after secretin alone. The results are discussed with respect to a possible influence of both lectins on the interaction of CCK or cerulein with the CCK-A receptor.

  3. Pharmacological properties of lorglumide as a member of a new class of cholecystokinin antagonists.

    PubMed

    Makovec, F; Bani, M; Cereda, R; Chisté, R; Pacini, M A; Revel, L; Rovati, L A; Rovati, L C; Setnikar, I

    1987-11-01

    Derivatives of 5-(dipentylamino)-5-oxo-pentanoic acid are a new class of non-peptide cholecystokinin (CCK) antagonists. The most potent compound, D,L-4-(3,4-dichlorobenzoylamino)-5-(dipentylamino)-5-oxo-pen tanoic acid (lorglumide, CR 1409), has a great affinity for the pancreatic CCK receptors and is a competitive, specific and potent CCK antagonist on the smooth muscles of the gall bladder and ileum of the guinea pig and on the CCK-induced amylase secretion of isolated pancreatic acini. In vivo lorglumide antagonizes the contraction of the gall bladder of the guinea pig and of the dog provoked by i.v. CCK-8 or ceruletide (caerulein). It antagonizes the satiety effect of CCK-8 in the rat and is protective against ceruletide-, taurocholate- and diet-induced pancreatitis. Lorglumide is therefore a useful pharmacological tool to study the functions of CCK. For its pharmacological properties, its relatively low toxicity and because it is active also after oral administration, lorglumide is a candidate for diagnostic or therapeutic use in man when an involvement of CCK is suspected.

  4. Antispasmodic activity on the gallbladder of the mouse of CR 1409 (lorglumide) a potent antagonist of peripheral CCK.

    PubMed

    Makovec, F; Bani, M; Cereda, R; Chistè, R; Pacini, M A; Revel, L; Rovati, L C

    1987-01-01

    Cholecystokinin (CCK) is a hormonal regulator of the motility of the gallbladder. CCK-8, i.e. the biologically active C-terminal octapeptide of the hormone, elicits contraction and emptying of the gallbladder. Endogenous CCK released by egg yolk or fatty acids in the duodenum gives the same results. CR 1409 (lorglumide), a glutaramic acid derivative with peripheric competitive CCK-antagonistic activity, was evaluated in comparison with proglumide (the model CCK-receptor antagonist) and other conventional antispasmodic drugs, for their ability to inhibit the emptying of the gallbladder induced in mice by CCK-8 or by lyophylized egg yolk. CR 1409 (1-10 mg/kg) prevented dose-dependently the emptying of the gallbladder in both experimental models; proglumide exhibited a comparable activity at much higher doses (200-800 mg/kg). On the contrary the anticholinergic drug atropine, the calcium-antagonist nifedipine, and the phosphodiesterase inhibitor papaverine were almost ineffective. The present data support the hypothesis that the effects of CCK on gallbladder motility are mediated by a CCK-dependent specific mechanism.

  5. CCK-5: sequence analysis of a small cholecystokinin from canine brain and intestine.

    PubMed

    Shively, J; Reeve, J R; Eysselein, V E; Ben-Avram, C; Vigna, S R; Walsh, J H

    1987-02-01

    The purpose of this study is to purify and to characterize chemically cholecystokinin (CCK)-like peptides present in brain and gut extracts that elute from gel filtration after the octapeptide. Canine small intestinal mucosa and brain were boiled in water and then extracted in cold trifluoroacetic acid, and cholecystokinin-like immunoreactivity was determined by carboxyl-terminal specific radioimmunoassay. Gel permeation chromatography on Sephadex G-50 revealed a form of CCK apparently smaller than CCK-8. This peptide was purified by immunoaffinity chromatography and three successive reverse phase high-performance liquid chromatography steps. Microsequence analysis showed that the amino terminal primary sequence of this small CCK was Gly-Trp-Met-Asp. Immunochemical and chromatographic analysis indicated that the carboxyl-terminal residue was Phe-NH2 and thus the full sequence is Gly-Trp-Met-Asp-Phe-NH2. An antibody that recognizes synthetic CCK-8, CCK-5, and CCK-4 equally did not reveal the presence of significant amounts of CCK-4. These results indicate that CCK-5 is the major CCK form smaller than the octapeptide present in brain (19% of total CCK immunoreactivity) and small intestine (7% of total). This finding, coupled with the demonstration by others that CCK-5 interacts with high-affinity brain CCK receptors, indicates that CCK-5 may play a physiological role in brain function.

  6. A high-fat diet attenuates the central response to within-meal satiation signals and modifies the receptor expression of vagal afferents in mice.

    PubMed

    Nefti, Wahiba; Chaumontet, Catherine; Fromentin, Gilles; Tomé, Daniel; Darcel, Nicolas

    2009-06-01

    During digestion, macronutrients are sensed within the small intestine. This sensory process is dependent upon the action of gut mediators, such as cholecystokinin (CCK) or serotonin (5-HT), on vagal afferents that, in turn, convey peripheral information to the brain to influence the control of food intake. Recent studies have suggested that dietary conditions alter vagal sensitivity to CCK and 5-HT. This phenomenon may be of importance to the onset of eating disorders. The aim of the present study was thus to investigate the effects of subjecting mice to 15 days of either an HF diet (30% fat, 54% carbohydrate) or an NF diet (10% fat, 74% carbohydrate) on 1) daily and short-term food intake, 2) vagal sensitivity to peripheral anorectic factors and macronutrient loads, and 3) vagal afferent neuron receptor expression. The results indicated that compared with an NF diet, and while increasing food intake and body weight gain, an HF diet altered the short-term response to CCK-8 and intragastric macronutrient loads, while decreasing vagal activation by CCK-8 and modifying the receptor expression of vagal neurons. These findings, therefore, suggest that dietary intervention effect on food intake could be linked to changes in vagal afferent receptor profiles.

  7. Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

    PubMed Central

    Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

    2016-01-01

    To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation. PMID:26727026

  8. Cholecystokinin plays a novel protective role in diabetic kidney through anti-inflammatory actions on macrophage: anti-inflammatory effect of cholecystokinin.

    PubMed

    Miyamoto, Satoshi; Shikata, Kenichi; Miyasaka, Kyoko; Okada, Shinichi; Sasaki, Motofumi; Kodera, Ryo; Hirota, Daisho; Kajitani, Nobuo; Takatsuka, Tetsuharu; Kataoka, Hitomi Usui; Nishishita, Shingo; Sato, Chikage; Funakoshi, Akihiro; Nishimori, Hisakazu; Uchida, Haruhito Adam; Ogawa, Daisuke; Makino, Hirofumi

    2012-04-01

    Inflammatory process is involved in the pathogenesis of diabetic nephropathy. In this article, we show that cholecystokinin (CCK) is expressed in the kidney and exerts renoprotective effects through its anti-inflammatory actions. DNA microarray showed that CCK was upregulated in the kidney of diabetic wild-type (WT) mice but not in diabetic intracellular adhesion molecule-1 knockout mice. We induced diabetes in CCK-1 receptor (CCK-1R) and CCK-2R double-knockout (CCK-1R(-/-),-2R(-/-)) mice, and furthermore, we performed a bone marrow transplantation study using CCK-1R(-/-) mice to determine the role of CCK-1R on macrophages in the diabetic kidney. Diabetic CCK-1R(-/-),-2R(-/-) mice revealed enhanced albuminuria and inflammation in the kidney compared with diabetic WT mice. In addition, diabetic WT mice with CCK-1R(-/-) bone marrow-derived cells developed more albuminuria than diabetic CCK-1R(-/-) mice with WT bone marrow-derived cells. Administration of sulfated cholecystokinin octapeptide (CCK-8S) ameliorated albuminuria, podocyte loss, expression of proinflammatory genes, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, CCK-8S inhibited both expression of tumor necrosis factor-α and chemotaxis in cultured THP-1 cells. These results suggest that CCK suppresses the activation of macrophage and expression of proinflammatory genes in diabetic kidney. Our findings may provide a novel strategy of therapy for the early stage of diabetic nephropathy.

  9. Possible degradative process of cholecystokinin analogs in rabbit jejunum brush-border membrane vesicles.

    PubMed

    Su, Sheng-Fang; Amidon, Gordon L; Lee, Hye J

    2002-11-22

    Our recent work on the intestinal metabolism and absorption of cholecystokinin analogs, sulfated C-terminal octapeptide (CCK8; Asp-Tyr(SO(3)H)-Met-Gly-Trp-Met-Asp-Phe(NH(2)) = DY(SO(3)H)MGWMDF(NH(2))) and tetrapeptide (CCK4; Trp-Met-Asp-Phe(NH(2)) = WMDF(NH(2))), was extended to investigate the degradative process of these analogs using rabbit jejunum brush-border membrane vesicles and to find a better enzyme-inhibitor system for intestinal absorption of peptide drugs. Various enzyme inhibitors and a lower pH buffer were applied to discover the major enzyme(s) involved in each process. Metabolic pathways showing degradative processes were proposed for both analogs. The major cleavage site occurs at the W(1)-M(2) for CCK4. At least three metabolic pathways occur independently for CCK8 and appear at peptides bonds between G(4)-W(5), M(6)-D(7), and D(7)-F(NH(2))(8). Many different enzymes of aminopeptidase, endopeptidase, angiotensin-converting enzyme, metalloenzyme, and others were involved in each process. Identification of more specific yet safe enzyme inhibitors and co-administration of various these inhibitors may lead to further enhancement in intestinal peptide absorption when administered orally.

  10. Moesin Expression Is Associated with Glioblastoma Cell Proliferation and Invasion.

    PubMed

    Wang, Qing; Lu, Xiaojie; Zhao, Shidi; Pang, Mingzhi; Wu, Xuechao; Wu, Heng; Hoffman, Robert M; Yang, Zhijian; Zhang, Yan

    2017-05-01

    To investigate the effect of moesin expression on cell proliferaton and invasion of human glioblastoma cell lines in vitro. Glioblastoma LN229 and U87 cells were transfected with the H4645-plenti-EGFP-moesin expression vector for moesin up-regulation. Moesin and β-catenin expression levels in the transfected cells were analyzed by real-time polymerase chain reaction (PCR) and Western blotting. Cell proliferation was measured using the CCK8 assay. Cell invasion and migration ability were assessed using a transwell cell invasion and wound-healing assay. Moesin mRNA and protein expression were significantly increased in the two transfected LN229-H4645 and U87-H4645 cell lines. β-catenin expression was increased by moesin up-regulation in the transfected LN229-H4645 and U87-H4645 cell lines. The CCK-8 assay revealed that up-regulating moesin results in a significant increase in glioblastoma cell proliferation. Glioblastoma cell invasion and migration were increased by moesin up-regulation. Up-regulation of moesin expression in glioblastoma cells correlated with increases in cell proliferation, invasion and migration, suggesting moesin's role in glioblastoma progression. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  11. Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques

    NASA Astrophysics Data System (ADS)

    Wang, Huiying; Chen, Tongsheng; Sun, Lei

    2008-02-01

    Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

  12. Suppression of RNA interference on expression of c-myc of SKOV3 ovarian carcinoma cell line.

    PubMed

    Ai, Z-H; Wang, J; Xu, Y-L; Zhu, X-L; Teng, Y-C

    2013-11-01

    To investigate suppression of RNA interference (RNAi) on expression of c-myc of SKOV3 ovarian carcinoma cell line. The c-myc -siRNA was designed and synthesized, then transfected to SKOV3 ovarian carcinoma cell lines. The cell lines were divided into four groups, including the blank control group, the siRNA transfection group, the mock transfection group and the negative control group. The expression level of c-myc mRNA and protein were detected by RT-PCR and Western blotting, respectively. The growth and proliferation of SKOV3 ovarian carcinoma cell lines were observed with CCK-8 assay. After transfected with c-myc -siRNA, the expression level of c-myc mRNA and protein were down-regulated, the growth and proliferation of SKOV3 ovarian carcinoma cell line were inhibited in the siRNA transfection group. There were significant differences between the siRNA transfection group and the blank control group (p < 0.05). The silencing efficiency was 77.78%, the protein suppression rate was 67.78%, and the inhibition ratio was 56.35% by CCK-8 assay in siRNA transfection group. The down-regulation of c-myc expression of SKOV3 ovarian carcinoma cell line by c-myc -siRNA can lead to the suppression of cancer cell proliferation. The small interfering RNAs technique can inhibit the proliferation of carcinoma cell by oncogene silencing.

  13. Synergistic antitumor effect of puerarin combined with 5-fluorouracil on gastric carcinoma

    PubMed Central

    GUO, XU-FENG; YANG, ZI-RONG; WANG, JUN; LEI, XIAO-FEI; LV, XIAO-GUANG; DONG, WEI-GUO

    2015-01-01

    Combination chemotherapy is a crucial method in the treatment of gastric cancer. The aim of the present study was to investigate the inhibitory effects of puerarin and 5-fluorouracil (5-FU) on BGC-823 gastric cancer cells in vitro and in vivo. The in vitro growth inhibition of puerarin or 5-FU alone or combined on BGC-823 cells was determined using a cell counting kit 8 (CCK-8) on living cells. Apoptotic morphological features and proteins expression levels were detected by Hoechst 33258 staining, an Annexin V/propidium iodide apoptosis kit and western blot analysis, respectively. Tumor xenografts were established in nude mice and the inhibitory effects and side effects were detected. Results of the CCK-8, Hoechst 33258 staining and flow cytometry revealed that the combined treatment was more effective than the separate treatments. The tumor volume was 90.65% of that of the controls and the mean tumor weight was only 0.125 g at the end of the experiment in the combination group compared with the control group (0.822 g). In addition, it was determined that liver and renal toxicity did not increase in combined treatment. These findings showed that puerarin and 5-FU produced a significant synergic effect on gastric cancer cells, while there was no increase in side effects. PMID:25434307

  14. Development and preclinical studies of (64)Cu-NOTA-pertuzumab F(ab')2 for imaging changes in tumor HER2 expression associated with response to trastuzumab by PET/CT.

    PubMed

    Lam, Karen; Chan, Conrad; Reilly, Raymond M

    2017-01-01

    We previously reported that microSPECT/CT imaging with (111)In-labeled pertuzumab detected decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to PET/CT by constructing F(ab')2 of pertuzumab modified with NOTA chelators for complexing (64)Cu. The effect of the administered mass (5-200 µg) of (64)Cu-NOTA-pertuzumab F(ab')2 was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian cancer xenografts. Biodistribution studies were performed in non-tumor bearing Balb/c mice to predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no significant effects of the administered mass of (64)Cu-NOTA-pertuzumab F(ab')2 on tumor or normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to (111)In-labeled pertuzumab. MicroPET/CT images revealed specific tumor uptake of (64)Cu-NOTA-pertuzumab F(ab')2 at 24 or 48 h post-injection in mice with SK-OV-3 tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of (64)Cu-NOTA-pertuzumab F(ab')2 in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with (64)Cu-NOTA-pertuzumab F(ab')2 detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to (111)In-labeled pertuzumab.

  15. Targeted actinium-225 in vivo generators for therapy of ovarian cancer.

    PubMed

    Borchardt, Paul E; Yuan, Rui R; Miederer, Matthias; McDevitt, Michael R; Scheinberg, David A

    2003-08-15

    Advanced ovarian cancer is largely incurable, but initially it is frequently confined to the i.p. space. We explored i.p. radioimmunotherapy in a mouse model of human ovarian cancer. Use of a targeted actinium-225 ((225)Ac) in vivo generator of alpha particles exploits the extreme, selective cytotoxicity of alpha particles, while providing a feasible half-life to enable delivery to tumor. (225)Ac chelated with 2-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid was conjugated to trastuzumab, an anti-HER-2/neu antibody. The radioimmunoconjugate was tested for immunoreactivity, internalization, and cytotoxicity using a human ovarian carcinoma cell line, SKOV3. (225)Ac-labeled trastuzumab retained immunoreactivity (50-90%), rapidly internalized into cells (50% at 2 h), and had an ED(50) of 1.3 nCi/ml after 4 days of incubation in vitro. i.p. administered (225)Ac- or (111)In-labeled trastuzumab behaved similarly with high tumor uptake [56-60% injected dose per gram (% ID/g) at 4 h, which increased to 65-70% ID/g at 24 h]. Tumor uptake was 3-5-fold higher than liver and spleen, the normal organs with the highest uptake. i.v. administration of (111)In-labeled trastuzumab produced slightly higher normal organ uptake compared with i.p.-administered (111)In-labeled trastuzumab. However, tumor uptake was low, 5%-26% ID/g. Therapy was examined with native trastuzumab and 220, 330, and 450 nCi of (225)Ac-labeled trastuzumab or (225)Ac-labeled control antibody at different dosing schedules. Therapy was initiated 9 days after tumor seeding. Groups of control mice and those administered native trastuzumab had median survivals of 33 and 37 or 44 days, respectively. Median survival was 52-126 days with (225)Ac-labeled trastuzumab at various doses and schedules, and 48-64 days for (225)Ac-labeled control the same schedules. Deaths from toxicity occurred with the highest activity levels. In conclusion, i.p. administration with a (225)Ac

  16. Development and evaluation of a new modular nanotransporter for drug delivery into nuclei of pathological cells expressing folate receptors.

    PubMed

    Slastnikova, Tatiana A; Rosenkranz, Andrey A; Khramtsov, Yuri V; Karyagina, Tatiana S; Ovechko, Sergey A; Sobolev, Alexander S

    2017-01-01

    Modular nanotransporters (MNTs) are artificial multifunctional systems designed to facilitate receptor-specific transport from the cell surface into the cell nucleus through inclusion of polypeptide domains for accomplishing receptor binding and internalization, as well as sequential endosomal escape and nuclear translocation. The objective of this study was to develop a new MNT targeted at folate receptors (FRs) for precise delivery of therapeutic cargo to the nuclei of FR-positive cells and to evaluate its potential, particularly for delivery of therapeutic agents (eg, the Auger electron emitter (111)In) into the nuclei of target cancer cells. A FR-targeted MNT was developed by site-specific derivatization of ligand-free MNT with maleimide-polyethylene glycol-folic acid. The ability of FR-targeted MNT to accumulate in target FR-expressing cells was evaluated using flow cytometry, and intracellular localization of this MNT was assessed using confocal laser scanning microscopy of cells. The cytotoxicity of the (111)In-labeled FR-targeted MNT was evaluated on HeLa and U87MG cancer cell lines expressing FR. In vivo micro-single-photon emission computed tomography/CT imaging and antitumor efficacy studies were performed with intratumoral injection of (111)In-labeled FR-targeted MNT in HeLa xenograft-bearing mice. The resulting FR-targeted MNT accumulated in FR-positive HeLa cancer cell lines specifically and demonstrated the ability to reach its target destination - the cell nuclei. (111)In-labeled FR-targeted MNT demonstrated efficient and specific FR-positive cancer cell eradication. A HeLa xenograft in vivo model revealed prolonged retention of (111)In delivered by FR-targeted MNT and significant tumor growth delay (up to 80% growth inhibition). The FR-targeted MNT met expectations of its ability to deliver active cargo into the nuclei of target FR-positive cells efficiently and specifically. As a result of this finding the new FR-targeted MNT approach warrants

  17. Roles of interleukin-9 in the growth and cholecystokinin-induced intracellular calcium signaling of cultured interstitial cells of Cajal.

    PubMed

    Gong, Yaoyao; Huang, Lei; Cheng, Wenfang; Li, Xueliang; Lu, Jia; Lin, Lin; Si, Xinmin

    2014-01-01

    Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract and loss of ICC is associated with many GI motility disorders. Previous studies have shown that ICC have the capacity to regenerate or restore, and several growth factors are critical to their growth, maintenance or regeneration. The present study aimed to investigate the roles of interleukin-9 (IL-9) in the growth, maintenance and pacemaker functions of cultured ICC. Here, we report that IL-9 promotes proliferation of ICC, and culturing ICC with IL-9 enhances cholecystokinin-8-induced Ca²⁺ transients, which is probably caused by facilitating maintenance of ICC functions under culture condition. We also show co-localizations of cholecystokinin-1 receptor and IL-9 receptor with c-kit by double-immunohistochemical labeling. In conclusion, IL-9 can promote ICC growth and help maintain ICC functions; IL-9 probably performs its functions via IL-9 receptors on ICC.

  18. ECL-cell histamine mobilization in conscious rats: effects of locally applied regulatory peptides, candidate neurotransmitters and inflammatory mediators.

    PubMed

    Norlén, P; Bernsand, M; Konagaya, T; Håkanson, R

    2001-12-01

    1. The ECL cells control gastric acid secretion by mobilizing histamine in response to circulating gastrin. In addition, the ECL cells are thought to operate under nervous control and to be influenced by local inflammatory processes. 2. The purpose of the present study was to monitor histamine mobilization from ECL cells in conscious rats in response to locally applied regulatory peptides, candidate neurotransmitters and inflammatory mediators. 3. Microdialysis probes were implanted in the submucosa of the acid-producing part of the rat stomach. Three days later, the agents to be tested were administered via the microdialysis probe and their effects on basal (48 h fast) and stimulated (intravenous infusion of gastrin-17, 3 nmol kg(-1) h(-1)) mobilization of ECL-cell histamine was monitored by continuous measurement of histamine in the perfusate (radioimmunoassay). 4. Locally administered gastrin-17 and sulfated cholecystokinin-8 mobilized histamine as did pituitary adenylate cyclase-activating peptide-27, vasoactive intestinal peptide, peptide YY, met-enkephalin, endothelin and noradrenaline, adrenaline and isoprenaline. 5. While gastrin, sulfated-cholecystokinin-8, met-enkephalin and isoprenaline induced a sustained elevation of the submucosal histamine concentration, endothelin, peptide YY, pituitary adenylate cyclase activating peptide, vasoactive intestinal peptide, noradrenaline and adrenaline induced a transient elevation. 6. Calcitonin gene-related peptide, galanin, somatostatin and the prostanoid misoprostol inhibited gastrin-stimulated histamine mobilization. 7. The gut hormones neurotensin and secretin and the neuropeptides gastrin-releasing peptide, neuropeptide Y and substance P failed to affect ECL-cell histamine mobilization, while motilin and neuromedin U-25 had weak stimulatory effects. Also acetylcholine, carbachol, serotonin and the amino acid neurotransmitters aspartate, gamma-aminobutyric acid, glutamate and glycine were inactive or weakly

  19. Blood clearance and organ distribution of intravenously administered polymethacrylic nanoparticles in mice

    SciTech Connect

    Rolland, A.; Collet, B.; Le Verge, R.; Toujas, L. )

    1989-06-01

    Polymethacrylic nanospheres (mean diameter 0.25-0.30 microns), prepared by aqueous emulsion copolymerization, were developed as a new site-specific drug delivery system. The nanoparticles were labeled either with indium-111 or iodine-125, and after a single iv injection of labeled particles into mice, their blood clearance and organ distribution were analyzed. A rapid clearance of {sup 111}In-labeled nanoparticles from the blood circulation to the mononuclear phagocyte system (MPS) was visualized using external scintigraphic imaging. From 10 to 60 min, radioactivity measurements in blood and organs (liver, spleen, kidneys, lungs, heart) showed that the {sup 125}I-labeled nanospheres were rapidly removed from the bloodstream (distribution half-life approximately 3-5 min) and mainly deposited in the liver (60% of the administered dose, 10 min after administration). Up to 1 h, radioactivity in heart and lungs remained insignificant, while in the kidneys, radioactivity levels increased from 8 to 11%.

  20. Imaging of platelets in right-sided extracardiac conduits in humans

    SciTech Connect

    Agarwal, K.C.; Wahner, H.W.; Dewanjee, M.K.; Fuster, V.; Puga, F.J.; Danielson, G.K.; Chesebro, J.H.; Feldt, R.H.

    1982-04-01

    As a connection between the systemic venous ventricle and the pulmonary artery, valved Dacron extracardiac conduits have remarkably influenced the surgical approach to many complex congenital heart defects. Obstruction of the conduit, however, can reduce the long-term effectiveness of this corrective procedure. In addition to stenosis of the porcine valve, formation of thick fibrous neointima plays a major role in the pathogenesis of conduit obstruction. The purpose of this study was to determine whether platelet deposition could be demonstrated in these conduits by external imaging with /sup 111/In-labeled autologous platelets. After injection of labeled platelets either immediately after operation or on the fifth to eighth postoperative day, imaging was performed by standard procedures. Eight of nine patients had platelet accumulation in the conduit, and treatment with aspirin and dipyridamole caused no recognizable change in platelet deposition. This study demonstrates the feasibility of imaging platelet deposition in Dacron conduits and shows that the pattern of deposition varies with time.

  1. Pulmonary accumulation of polymorphonuclear leukocytes in the adult respiratory distress syndrome

    SciTech Connect

    Powe, J.E.; Short, A.; Sibbald, W.J.; Driedger, A.A.

    1982-11-01

    The polymorphonuclear leukocyte (PMN) plays an integral role in the development of permeability pulmonary edema associated with the adult respiratory distress syndrome (ARDS). This report describes 3 patients with ARDS secondary to systemic sepsis who demonstrated an abnormal diffuse accumulation of Indium (/sup 111/In)-labeled PMNs in their lungs, without concomitant clinical or laboratory evidence of a primary chest infection. In one patient, the accumulation of the pulmonary activity during an initial pass suggested that this observation was related to diffuse leukoaggregation within the pulmonary microvasculature. A 4th patient with ARDS was on high-dose corticosteroids at the time of a similar study, and showed no pulmonary accumulation of PMNs, suggesting a possible reason for the reported beneficial effect of corticosteroids in human ARDS.

  2. /sup 111/In platelet scintigraphy in cerebrovascular disease

    SciTech Connect

    Powers, W.J.; Siegel, B.A.; Davis, H.H.; Mathias, C.J.; Clark, H.B.; Welch, M.J.

    1982-09-01

    We obtained scintigraphic images of the neck from 100 patients with suspected cerebrovascular disease after injecting /sup 111/In-labeled autologous platelets. One or more focuses of increased activity, implying local platelet accumulation, were seen along the course of the cervical carotid arteries in 52 patients. In 64 patients, there was a highly significant correlation between the results of scintigraphy and carotid arteriography (p . 10(6)). There was no significant correlation between the scintigraphic findings and the previous or subsequent occurrence of transient ischemic attack or cerebral infarction in the carotid circulation. These data suggest that factors other than the simple formation of platlet thrombi in the cervical carotid arteries are of primary importance in the pathogenesis of stroke.

  3. In vivo dissolution measurement with indium-111 summation peak ratios

    SciTech Connect

    Jay, M.; Woodward, M.A.; Brouwer, K.R.

    1985-10-01

    Dissolution of (/sup 111/In)labeled tablets was measured in vivo in a totally noninvasive manner by using a modification of the perturbed angular correlation technique known as the summation peak ratio method. This method, which requires the incorporation of only 10-12 microCi into the dosage form, provided reliable dissolution data after oral administration of (/sup 111/In)lactose tablets. These results were supported by in vitro experiments which demonstrated that the dissolution rate as measured by the summation peak ratio method was in close agreement with the dissolution rate of salicylic acid in a (/sup 111/In)salicylic acid tablet. The method has the advantages of using only one detector, thereby avoiding the need for complex coincidence counting systems, requiring less radioactivity, and being potentially applicable to a gamma camera imaging system.

  4. Effect of acetylation on monoclonal antibody ZCE-025 Fab': Distribution in normal and tumor-bearing mice

    SciTech Connect

    Tarburton, J.P.; Halpern, S.E.; Hagan, P.L.; Sudora, E.; Chen, A.; Fridman, D.M.; Pfaff, A.E. )

    1990-04-01

    Studies were performed to determine in vitro and in vivo effects of acetylation on Fab' fragments of ZCE-025, a monoclonal anti-CEA antibody. Isoelectric focusing revealed a drop in isoelectric point of 1.7 pI units following acetylation. Biodistribution studies of acetylated and nonacetylated (111In)Fab' were performed in normal BALB/c mice and in nude mice bearing the T-380 CEA-producing human colon tumor. The acetylated fragments remained in the vascular compartment longer and had significantly diminished renal uptake of 111In compared to controls. While acetylation itself effected a 50% drop in immunoreactivity, tumor uptake of the acetylated and nonacetylated 111In-labeled Fab' fragments was comparable, with the exception of one data point, through 72 h.

  5. A motility test of leukocytes under agar.

    PubMed

    Goedemans, W T; de Jong, M M

    1985-01-01

    A migration test under agar for leukocytes was developed. Leukocytes moved quite a distance under anaerobic Blood Agar Base (blood agar), a Gibco product. Migration on stained and coloured plates was visualized by projection with a profile projector, making the use of a light microscope superfluous. A migration index was defined. Reproducibility was good enough to allow paired comparisons of leukocyte populations subjected to different treatments. Migration was the result of spontaneous and chemotactically directed migration. Cell-labelling complexes as 111In-oxinate and 111In-tropolonate--ligand concentration 3.5 micrograms/mL in the ultimate cell preparation--did not affect leukocyte migration. 111In-pyrithionate (mercapto pyridine-N-oxide) significantly impaired cell motility. The motility test described could be used as retrospective analysis in abscess localization studies using 111In labelled leukocytes.

  6. Standardisation of tracer and type of meal in the evaluation of gastric emptying and colon transit rates in normal beagle dogs.

    PubMed

    Dormehl, I C; Pilloy, W; Jordaan, J H; Jacobs, D J; du Plessis, M

    1984-01-01

    The transit 111In-labelled polymer beads of different particle sizes in the stomach and colon of five healthy Beagle dogs was monitored after intake of different kinds of meal by a gamma camera and data processor system. The various meals studied were a solid balanced type of dog food mixed with milk, pure milk, and finally a meal of raw meat. The polymer beads carried triethylenetetramine type functions which efficiently chelated 111In+3 from an aqueous solution of InCl3. These labelled beads were well mixed into the food before being offered to the dogs. Gastric emptying curves from which half-times of emptying (T 1/2) could be obtained are presented and statistically compared. Good quality images with quantification of the colon transit rate also became possible. It appeared that standardisation of the tracer and of the meal is compulsory for relevant and reproducible results.

  7. Spinal dural enhancement on magnetic resonance imaging associated with spontaneous intracranial hypotension. Report of three cases and review of the literature.

    PubMed

    Moayeri, N N; Henson, J W; Schaefer, P W; Zervas, N T

    1998-05-01

    This report offers a description of typical changes seen on gadolinium-enhanced magnetic resonance (MR) imaging of the entire spine that indicate spontaneous intracranial hypotension (SIH). To the authors' knowledge, this is the first report of its kind. They describe three cases of SIH that were accompanied by dural enhancement throughout the neuraxis on imaging, with the evolution of associated subdural and epidural fluid collections in the spine. Recognition of this disorder is important to be able to distinguish it from an infectious or neoplastic process in which surgical intervention might be warranted. Evaluation using gadolinium-enhanced cranial and spinal MR imaging in patients with postural headaches and an (111)In-labeled cerebrospinal fluid leak study are discussed. Treatment with an epidural blood patch is shown to be particularly effective, with resolution of the radiological and clinical findings.

  8. An in vitro method for assessing biomaterial-associated platelet activation.

    PubMed

    LaFayette, Nathan G; Skrzypchak, Amy M; Merz, Scott; Bartlett, Robert H; Annich, Gail M

    2007-01-01

    The development of a nonthrombogenic artificial surface for use with indwelling sensors or catheters remains an elusive goal despite decades of ongoing research. In vivo studies are both labor intensive and costly, and are therefore an inefficient way to rapidly screen possible surface materials. The following in vitro model used glass, polyvinyl chloride (PVC), and polypropylene test tubes incubated with 111In-labeled rabbit platelets and illustrated that, despite equivalent platelet count and function, platelet adhesion was greatest on glass (n = 13), with PVC (n = 17) at 67 +/- 8% and polypropylene (n = 13) at 43 +/- 5% when compared with glass. Extrapolating this method by coating test tubes with new, nonthrombogenic materials is a quick and reliable way to screen material before embarking upon more lengthy in vivo animal studies.

  9. Role of ProstaScint for brachytherapy in localized prostate adenocarcinoma.

    PubMed

    Ellis, Rodney J; Kim, Edward; Foor, Ryan

    2004-07-01

    ProstaScint (CYT-356 or capromab pendetide, Cytogen) is an 111In-labeled monoclonal mouse antibody specific for prostate-specific membrane antigen, a prostate transmembrane glycoprotein that is upregulated in prostate adenocarcinoma. ProstaScint scans are US Food and Drug Administration approved for pretreatment evaluation of metastatic disease in high-risk patients. They are also approved for post-prostatectomy assessment of recurrent disease in patients with a rising prostate-specific antigen level. This review explores the literature on ProstaScint and its use in guiding the treatment of prostate cancer. A novel technique for identifying areas of cancer within the prostate using ProstaScint images fused with pelvic computed tomography scans is also described. The identification of areas of high antibody signal provides targets for radiotherapeutic dose escalation, with the overall goals of improving treatment outcome while preserving adjacent tissue structures and decreasing treatment morbidity.

  10. 99mTc-HMPAO-labeled WBC scan for the diagnosis of chronic recurrent multifocal osteomyelitis.

    PubMed

    Dailey, Tyler A; Berven, Michael D; Vroman, Penny J

    2014-12-01

    The goal of this teaching case study is to demonstrate the novel use of (99m)Tc-HMPAO-labeled white blood cells (WBCs) in diagnosing chronic recurrent multifocal osteomyelitis (CRMO). (99m)Tc-HMPAO-labeled white blood cells were utilized to diagnose CRMO. Classically, CRMO is evaluated scintigraphically using (99m)Tc-methylene diphosphonate/hydroxymethylene diphosphonate. (99m)Tc-HMPAO-labeled WBCs were chosen over (111)In-labeled WBCs because of the former's improved imaging characteristics and decreased radiation dose. (99m)Tc-HMPAO-labeled WBCs were successful in diagnosing CRMO. (99m)Tc-HMPAO-labeled WBC scanning is specific for the diagnosis of CRMO. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  11. Spleen function and platelet kinetics.

    PubMed Central

    Klonizakis, I; Peters, A M; Fitzpatrick, M L; Kensett, M J; Lewis, S M; Lavender, J P

    1981-01-01

    In patients suffering from various platelet abnormalities, quantitative scanning after injection of indium-111 (111In) labelled platelets showed three different patterns of platelet destruction and distribution. In patients with a normal platelet life span but with evidence of increased splenic pooling, the spleen tended to be the main site of destruction. In patients with a moderately reduced platelet life span, the distribution of destruction in the system and variable destruction in the marrow. However, because of its rapidity this destruction was difficult to quantify, and it was difficult in these cases to distinguish reliably between spleen pool, sequestration, and destruction. Destruction of platelets on the liver appeared to be unimportant in all three groups. 111In, because of its physical characteristics, is preferable to chromium-51 as a platelet label in the assessment of abnormal platelet kinetics. Images PMID:7240424

  12. The Cyclotron radionuclide program at King Faisal Specialist Hospital and Research Centre

    NASA Astrophysics Data System (ADS)

    Hupf, Homer B.; Tischer, Stephen D.; Al-Watban, Farouk

    1985-05-01

    The King Faisal Specialist Hospital and Research Centre Cyclotron is being used to produce radionuclides for nuclear medicine, short-lived positron emitters for positron emission tomography (PET) studies, neutrons for therapy and biological research. Radiopharmaceuticals for planar imaging at King Faisal Specialist Hospital and other hospitals in Saudi Arabia include thallous-201 chloride, gallium-67 citrate, sodium iodide 123I capsules, 123I orthoiodohippurate and 81mKr generators. Products from short-lived positron emitters such as 18F fluordeoxyglucose, 11C methionine, 15O carbon dioxide and 63Zn hematoporphyrin are prepared and used on site for physiological studies in a PET program. Several patients have been treated with neutron therapy and a program for studying neutron radiation effects on cells is underway. Radiopharmaceutical products under development include 111In-labelled monoclonal antibodies for specific tumor detection, 11C methylglucose for metabolic studies and 11C putrescine for tumor localization.

  13. Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response.

    PubMed

    Noe, Stephanie M; Green, Mark A; HogenEsch, Harm; Hem, Stanley L

    2010-04-30

    The relationship between depot formation and immunopotentiation was studied by comparing the retention of antigen at the inoculation site with antibody production in rats. A model (111)In-labeled alpha casein (IDCAS) antigen was formulated into four vaccines: IDCAS adsorbed onto either aluminum hydroxide adjuvant (AH) or aluminum phosphate adjuvant (AP); non-adsorbed IDCAS with phosphate-treated AP (PTAP); and IDCAS solution. Gamma scintigraphy showed the order of retention following subcutaneous administration to be: AH adsorbed>AP adsorbed>non-adsorbed with PTAP=solution. The antibody titers followed the order: non-adsorbed with PTAP=AP adsorbed>AH adsorbed>solution. The presence of an aluminum-containing adjuvant was essential for immunopotentiation, but retention of the antigen at the inoculation site was not required. (c) 2010 Elsevier Ltd. All rights reserved.

  14. Triple Therapy of HER2(+) Cancer Using Radiolabeled Multifunctional Iron Oxide Nanoparticles and Alternating Magnetic Field.

    PubMed

    Zolata, Hamidreza; Afarideh, Hossein; Davani, Fereydoun Abbasi

    2016-11-01

    By using radio-labeled multifunctional superparamagnetic iron oxide nanoparticles (SPIONs) and an alternating magnetic field (AMF), we carried out targeted hyperthermia, drug delivery, radio-immunotherapy (RIT), and controlled chemotherapy of cancer tumors. We synthesized and characterized Indium-111-labeled, Trastuzumab and Doxorubicin (DOX)-conjugated APTES-PEG-coated SPIONs in our previous work. Then, we evaluated their capability in SPECT/MRI (single photon emission computed tomography/magnetic resonance imaging) dual modal molecular imaging, targeting, and controlled release. In this research, AMF was introduced to evaluate therapeutic effects of magnetic hyperthermia on radionuclide-chemo therapy of HER2(+) cells and tumor (HER2(+))-bearing mice. In vitro and in vivo experiments using synthesized complex were repeated under an AMF (f: 100 KHz, H: 280 Gs). Instead of an intra-tumor injection in most hyperthermia experiments, SPIONs were injected to the tail vein, based on our delivery strategies. For magnetic delivery, we held a permanent Nd-B-Fe magnet near the tumor region. The results showed that simultaneous magnetic hyperthermia enhanced SKBR3 cancer cells, killing by 24%, 28%, 33%, and 80% at 48 hours post-treatment for treated cells with (1) bare SPIONs; (2) antibody-conjugated, DOX-free, surface-modified SPIONs; (3) (111)In-labeled, antibody-conjugated surface-modified SPIONs; and (4) (111)In-labeled, antibody- and DOX-conjugated surface-modified SPIONs, respectively. Moreover, tumor volume inhibitory rate was 85% after a 28 day period of treatment. By using this method, multimodal imaging-guided, targeted hyperthermia, RIT, and controlled chemotherapy could be achievable in the near future.

  15. Cardiac and vascular imaging with labeled platelets and leukocytes

    SciTech Connect

    Dewanjee, M.K.

    1984-07-01

    The contribution of platelets in atherosclerosis and thrombosis in animal models and in clinical studies has been quantified with 111In-platelet scintigraphy. New in vitro quantitative techniques have been developed using 111In-labeled platelets to determine the number of adherent platelets on deendothelialized surfaces of damaged vessel walls and synthetic vascular grafts. In vivo imaging techniques are semi-quantitative in nature; in these studies 111In radioactivity on thrombotic vessels or graft surfaces of iliac, femoral, or popliteal arteries is compared with contralateral vessels. Background 111In radioactivity in the circulating blood pool of venous and capillary networks and radioactivity in marrow decreases the sensitivity of these techniques. Subtraction of blood pool radioactivity with 99mTc-labeled autologous red cells and calculation of 111In radioactivity associated with platelet thrombus on vessel walls also have been performed for coronary, carotid, and femoral arteries. Although platelet concentrates are used frequently after open heart surgery (one to six per patient), consumption of platelets in the artificial lung or oxygenator, lysis of platelets during pumping, and suction of blood only recently have been quantified with the use of 111In-labeled platelets. These studies also demonstrated far less trauma to platelets with the use of a membrane rather than a bubble oxygenator. Further reduction in platelet consumption and trauma was observed with the use of prostacyclin, a short-acting drug with significant beneficial effect on platelet thrombus reduction and disaggregation of aggregated platelets. The role of polymorphonuclear leukocytes in inflammation, infection and myocardial infarction, and in vivo evaluation with 111In-leukocyte scintigraphy in animals and humans has been described.

  16. Trastuzumab labeled to high specific activity with ¹¹¹In by conjugation to G4 PAMAM dendrimers derivatized with multiple DTPA chelators exhibits increased cytotoxic potency on HER2-positive breast cancer cells.

    PubMed

    Chan, Conrad; Cai, Zhongli; Reilly, Raymond M

    2013-08-01

    To conjugate trastuzumab with/without NLS peptides to G4 PAMAM dendrimers derivatized with DTPA and determine the specific radioactivity (SA) for (111)In labeling, HER2 immunoreactivity and cytotoxicity on breast cancer (BC) cells. G4 dendrimers were reacted with DTPA then conjugated through a thiol to maleimide-derivatized trastuzumab. The SA achievable was determined by incubating 2 to 20 μg with 60 MBq of (111)In. HER2 immunoreactivity, internalization and nuclear importation were measured. The effect of (111)In-DTPA-G4-trastuzumab (5.9 MBq/μg) on the clonogenic survival (CS) of SK-Br-3 or MDA-MB-231 cells with high or low HER2 density, respectively was compared to (111)In-DTPA-NLS-trastuzumab (0.5 MBq/μg). DNA double-strand breaks (DSBs) were measured. DTPA-G4-trastuzumab was labeled with (111)In to a SA (23.6 MBq/μg) which was 100-fold higher than (111)In-DTPA-NLS-trastuzumab. (111)In-DTPA-G4-trastuzumab and (111)In-DTPA-G4-NLS-trastuzumab retained HER2 immunoreactivity and were internalized and imported into the nucleus of BC cells. G4-radioimmunoconjugates were 2-4 fold and 9-fold more cytotoxic to SK-Br-3 and MDA-MB-231 cells, respectively than (111)In-DTPA-NLS-trastuzumab which was associated with an increase in DNA DSBs. Conjugation of trastuzumab to G4 PAMAM dendrimers modified with 30 DTPA permitted high SA (111)In labeling which increased their cytotoxic potency for BC cells with high or low HER2 density.

  17. Low density lipoprotein receptor-independent hepatic uptake of a synthetic, cholesterol-scavenging lipoprotein: implications for the treatment of receptor-deficient atherosclerosis.

    PubMed Central

    Williams, K J; Vallabhajosula, S; Rahman, I U; Donnelly, T M; Parker, T S; Weinrauch, M; Goldsmith, S J

    1988-01-01

    The metabolism of infused 111In-labeled phospholipid liposomes was examined in Watanabe heritable hyperlipidemic (WHHL) rabbits, which lack low density lipoprotein (LDL) receptors, and in normal control rabbits. The half-times (t1/2) for clearance of 111In and excess phospholipid from plasma were 20.8 +/- 0.9 hr and 20.3 +/- 4.6 hr in WHHL and 20.0 +/- 0.8 hr and 19.6 +/- 2.2 hr in the normal rabbits (means +/- SEM; n = 4). By 6 hr postinfusion, the plasma concentration of unesterified cholesterol increased by 2.2 +/- 0.23 mmol/liter in WHHL and 2.1 +/- 0.04 mmol/liter in normal rabbits, presumably reflecting mobilization of tissue stores. Disappearance of excess plasma cholesterol was greater than 90% complete in both groups of rabbits by 70 hr postinfusion. By quantitative gamma camera imaging, hepatic trapping of 111In-labeled liposomes over time was indistinguishable between the two groups. At autopsy, the liver was the major organ of clearance, acquiring 22.0% +/- 1.7% (WHHL) and 16.8% +/- 1.0% (normal of total 111In. Aortic uptake of 111In was less than 0.02%. Thus, mobilization of cholesterol and hepatic uptake of phospholipid liposomes do not require LDL receptors. Because phospholipid infusions produce rapid substantial regression of atherosclerosis in genetically normal animals, our results suggest that phospholipid liposomes or triglyceride phospholipid emulsions (e.g., Intralipid) might reduce atherosclerosis in WHHL rabbits and in humans with familial hypercholesterolemia. PMID:3422421

  18. Indium-111-labeled autologous leukocyte imaging and fecal excretion. Comparison with conventional methods of assessment of inflammatory bowel disease

    SciTech Connect

    Leddin, D.J.; Paterson, W.G.; DaCosta, L.R.; Dinda, P.K.; Depew, W.T.; Markotich, J.; McKaigney, J.P.; Groll, A.; Beck, I.T.

    1987-04-01

    This study was designed to evaluate the role of /sup 111/In-labeled leukocyte imaging and fecal excretion in the assessment of inflammatory bowel disease. We compared these tests to various indices of disease activity in Crohn's disease, to Truelove's grading in ulcerative colitis, and to endoscopy, x-ray, and pathology in both diseases. Eleven controls, 16 patients with Crohn's disease, 13 with ulcerative colitis, and 3 with other types of acute bowel inflammation were studied (positive controls). Indium scanning was performed at 1, 4, and 24 hr. Fourteen of 16 patients with active Crohn's disease had positive scans but in only five was localization accurate. One patient had inactive ulcerative colitis, and the scan was negative. Of 12 patients with active ulcerative colitis, 10 had positive scans but disease localization was accurate in only four. Disease extent was correctly defined in 1 of the 3 Positive Controls. There was no significant difference in the accuracy of scanning at 1, 4, or 24 hr. /sup 111/In fecal excretion was significantly higher in patients with inflammatory bowel disease than in controls, and there was correlation between /sup 111/In fecal excretion and most of the indices of disease activity in Crohn's disease. In ulcerative colitis, /sup 111/In fecal excretion did not correlate with Truelove's grading but reflected colonoscopic assessment of severity. In conclusion, /sup 111/In-labeled leukocyte scanning lacks sensitivity with respect to disease extent, but fecal excretion of /sup 111/In correlates well with disease severity as determined by other methods.

  19. Comparison of autologous 111In-leukocytes, 18F-FDG, 11C-methionine, 11C-PK11195 and 68Ga-citrate for diagnostic nuclear imaging in a juvenile porcine haematogenous staphylococcus aureus osteomyelitis model

    PubMed Central

    Nielsen, Ole L; Afzelius, Pia; Bender, Dirk; Schønheyder, Henrik C; Leifsson, Páll S; Nielsen, Karin M; Larsen, Jytte O; Jensen, Svend B; Alstrup, Aage KO

    2015-01-01

    The aim of this study was to compare 111In-labeled leukocyte single-photon emission computed tomography (SPECT) and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) to PET with tracers that potentially could improve detection of osteomyelitis. We chose 11C-methionine, 11C-PK11195 and 68Ga-citrate and validated their diagnostic utility in a porcine haematogenous osteomyelitis model. Four juvenile 14-15 weeks old female pigs were scanned seven days after intra-arterial inoculation in the right femoral artery with a porcine strain of Staphylococcus aureus using a sequential scan protocol with 18F-FDG, 68Ga-citrate, 11C-methionine, 11C-PK11195, 99mTc-Nanocoll and 111In-labelled autologous leukocytes. This was followed by necropsy of the pigs and gross pathology, histopathology and microbial examination. The pigs developed a total of five osteomyelitis lesions, five lesions characterized as abscesses/cellulitis, arthritis in three joints and five enlarged lymph nodes. None of the tracers accumulated in joints with arthritis. By comparing the 10 infectious lesions, 18F-FDG accumulated in nine, 111In-leukocytes in eight, 11C-methionine in six, 68Ga-citrate in four and 11C-PK11195 accumulated in only one lesion. Overall, 18F-FDG PET was superior to 111In-leukocyte SPECT in marking infectious and proliferative, i.e. hyperplastic, lesions. However, leukocyte SPECT was performed as early scans, approximately 6 h after injection of the leukocytes, to match the requirements of the 18 h long scan protocol. 11C-methionine and possibly 68Ga-citrate may be useful for diagnosis of soft issue lesions. PMID:25973338

  20. Synthesis of DOTA-conjugated multimeric [Tyr3]octreotide peptides via a combination of Cu(I)-catalyzed "click" cycloaddition and thio acid/sulfonyl azide "sulfo-click" amidation and their in vivo evaluation.

    PubMed

    Yim, Cheng-Bin; Dijkgraaf, Ingrid; Merkx, Remco; Versluis, Cees; Eek, Annemarie; Mulder, Gwenn E; Rijkers, Dirk T S; Boerman, Otto C; Liskamp, Rob M J

    2010-05-27

    Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated monomeric, dimeric, and tetrameric [Tyr(3)]octreotide-based analogues as a tool for tumor imaging and/or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar cycloaddition ("click" reaction) between peptidic azides and dendrimer-derived alkynes and a subsequent metal-free introduction of DOTA via the thio acid/sulfonyl azide amidation ("sulfo-click" reaction). In a competitive binding assay using rat pancreatic AR42J tumor cells, the monomeric [Tyr(3)]octreotide conjugate displayed the highest binding affinity (IC(50) = 1.32 nM) followed by dimeric [Tyr(3)]octreotide (2.45 nM), [DOTA(0),Tyr(3)]octreotide (2.45 nM), and tetrameric [Tyr(3)]octreotide (14.0 nM). Biodistribution studies with BALB/c nude mice with subcutaneous AR42J tumors showed that the (111)In-labeled monomeric [Tyr(3)]octreotide conjugate had the highest tumor uptake (42.3 +/- 2.8 %ID/g) at 2 h p.i., which was better than [(111)In-DOTA(0),Tyr(3)]octreotide (19.5 +/- 4.8 %ID/g). The (111)In-labeled dimeric [Tyr(3)]octreotide conjugate showed a long tumor retention (25.3 +/- 5.9 %ID/g at 2 h p.i. and 12.1 +/- 1.3 %ID/g at 24 h p.i.). These promising results can be exploited for therapeutic applications.

  1. Preclinical evaluation of (111)In-DTPA-INCA-X anti-Ku70/Ku80 monoclonal antibody in prostate cancer.

    PubMed

    Evans-Axelsson, Susan; Vilhelmsson Timmermand, Oskar; Welinder, Charlotte; Borrebaeck, Carl Ak; Strand, Sven-Erik; Tran, Thuy A; Jansson, Bo; Bjartell, Anders

    2014-01-01

    The aim of this investigation was to assess the Ku70/Ku80 complex as a potential target for antibody imaging of prostate cancer. We evaluated the in vivo and ex vivo tumor targeting and biodistribution of the (111)In-labeled human internalizing antibody, INCA-X ((111)In-DTPA-INCA-X antibody), in NMRI-nude mice bearing human PC-3, PC-3M-Lu2 or DU145 xenografts. DTPA-conjugated, non-labeled antibody was pre-administered at different time-points followed by a single intravenous injection of (111)In-DTPA-INCA-X. At 48, 72 and 96 h post-injection, tissues were harvested, and the antibody distribution was determined by measuring radioactivity. Preclinical SPECT/CT imaging of mice with and without the predose was performed at 48 hours post-injection of labeled DTPA-INCA-X. Biodistribution of the labeled antibody showed enriched activity in tumor, spleen and liver. Animals pre-administered with DTPA-INCA-X showed increased tumor uptake and blood content of (111)In-DTPA-INCA-X with reduced splenic and liver uptake. The in vitro and in vivo data presented show that the (111)In-labeled INCA-X antibody is internalized into prostate cancer cells and by pre-administering non-labeled DTPA-INCA-X, we were able to significantly reduce the off target binding and increase the (111)In-DTPA-INCA-X mAb uptake in PC-3, PC-3M-Lu2 and DU145 xenografts. The results are encouraging and identifying the Ku70/Ku80 antigen as a target is worth further investigation for functional imaging of prostate cancer.

  2. Prevention of ischemia-induced myocardial platelet deposition by exogenous prostacyclin

    SciTech Connect

    Aherne, T.; Price, D.C.; Yee, E.S.; Hsieh, W.R.; Ebert, P.A.

    1986-07-01

    The antithrombotic effects of prostacyclin infusion on myocardial platelet deposition were studied in a canine model during and after global ischemia. Eleven isolated heart preparations were subjected to 1 hour of cardioplegic arrest under moderate hypothermia (27 to 28/sup 0/C), including a control group (n = 7) and a prostacyclin-treated group (n = 4). The hearts of four other dogs were continuously perfused for 180 minutes. Platelet deposition was measured at 15 minute intervals throughout the 3 hour study. Serial full-thickness myocardial biopsy specimens were analyzed for activity of /sup 111/In-labeled platelets with /sup 99m/Tc-labeled erythrocyte correction for tissue blood content. The pattern of platelet distribution was determined by scintiscans of each heart, taken with a gamma camera at the end of the 60 minute reperfusion period. Substantial myocardial platelet deposition was found in the control hearts after ischemia but not in the prostacyclin-treated group (p less than 0.05). Furthermore, prostacyclin infusion had a significant disaggregatory effect on intracoronary platelet deposits when the precardioplegic and postcardioplegic biopsy specimens were analyzed (p less than 0.05). Three hours of continuous perfusion did not increase tissue /sup 111/In-labeled platelet activity. Ex vivo images showed platelet deposition to be a diffuse patchy process with significantly more /sup 111/In activity in the endocardium than in the epicardium after global ischemia (p less than 0.05). These data show the potent antithrombotic properties of prostacyclin in preventing and disaggregating ischemia-induced intracoronary platelet deposition during and after cardioplegic arrest.

  3. Maximizing tumour exposure to anti-neuropilin-1 antibody requires saturation of non-tumour tissue antigenic sinks in mice

    PubMed Central

    Bumbaca, Daniela; Xiang, Hong; Boswell, C Andrew; Port, Ruediger E; Stainton, Shannon L; Mundo, Eduardo E; Ulufatu, Sheila; Bagri, Anil; Theil, Frank-Peter; Fielder, Paul J; Khawli, Leslie A; Shen, Ben-Quan

    2012-01-01

    BACKGROUND AND PURPOSE Neuropilin-1 (NRP1) is a VEGF receptor that is widely expressed in normal tissues and is involved in tumour angiogenesis. MNRP1685A is a rodent and primate cross-binding human monoclonal antibody against NRP1 that exhibits inhibition of tumour growth in NPR1-expressing preclinical models. However, widespread NRP1 expression in normal tissues may affect MNRP1685A tumour uptake. The objective of this study was to assess MNRP1685A biodistribution in tumour-bearing mice to understand the relationships between dose, non-tumour tissue uptake and tumour uptake. EXPERIMENTAL APPROACH Non-tumour-bearing mice were given unlabelled MNRP1685A at 10 mg·kg−1. Tumour-bearing mice were given 111In-labelled MNRP1685A along with increasing amounts of unlabelled antibody. Blood and tissues were collected from all animals to determine drug concentration (unlabelled) or radioactivity level (radiolabelled). Some animals were imaged using single photon emission computed tomography – X-ray computed tomography. KEY RESULTS MNRP1685A displayed faster serum clearance than pertuzumab, indicating that target binding affected MNRP1685A clearance. I.v. administration of 111In-labelled MNRP1685A to tumour-bearing mice yielded minimal radioactivity in the plasma and tumour, but high levels in the lungs and liver. Co-administration of unlabelled MNRP1685A with the radiolabelled antibody was able to competitively block lungs and liver radioactivity uptake in a dose-dependent manner while augmenting plasma and tumour radioactivity levels. CONCLUSIONS AND IMPLICATIONS These results indicate that saturation of non-tumour tissue uptake is required in order to achieve tumour uptake and acceptable exposure to antibody. Utilization of a rodent and primate cross-binding antibody allows for translation of these results to clinical settings. PMID:22074316

  4. Maximizing tumour exposure to anti-neuropilin-1 antibody requires saturation of non-tumour tissue antigenic sinks in mice.

    PubMed

    Bumbaca, Daniela; Xiang, Hong; Boswell, C Andrew; Port, Ruediger E; Stainton, Shannon L; Mundo, Eduardo E; Ulufatu, Sheila; Bagri, Anil; Theil, Frank-Peter; Fielder, Paul J; Khawli, Leslie A; Shen, Ben-Quan

    2012-05-01

    Neuropilin-1 (NRP1) is a VEGF receptor that is widely expressed in normal tissues and is involved in tumour angiogenesis. MNRP1685A is a rodent and primate cross-binding human monoclonal antibody against NRP1 that exhibits inhibition of tumour growth in NPR1-expressing preclinical models. However, widespread NRP1 expression in normal tissues may affect MNRP1685A tumour uptake. The objective of this study was to assess MNRP1685A biodistribution in tumour-bearing mice to understand the relationships between dose, non-tumour tissue uptake and tumour uptake. Non-tumour-bearing mice were given unlabelled MNRP1685A at 10 mg·kg(-1) . Tumour-bearing mice were given (111) In-labelled MNRP1685A along with increasing amounts of unlabelled antibody. Blood and tissues were collected from all animals to determine drug concentration (unlabelled) or radioactivity level (radiolabelled). Some animals were imaged using single photon emission computed tomography - X-ray computed tomography. MNRP1685A displayed faster serum clearance than pertuzumab, indicating that target binding affected MNRP1685A clearance. I.v. administration of (111) In-labelled MNRP1685A to tumour-bearing mice yielded minimal radioactivity in the plasma and tumour, but high levels in the lungs and liver. Co-administration of unlabelled MNRP1685A with the radiolabelled antibody was able to competitively block lungs and liver radioactivity uptake in a dose-dependent manner while augmenting plasma and tumour radioactivity levels. These results indicate that saturation of non-tumour tissue uptake is required in order to achieve tumour uptake and acceptable exposure to antibody. Utilization of a rodent and primate cross-binding antibody allows for translation of these results to clinical settings. © 2011 Genentech Inc. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  5. Role of platelet-activating factor in polymorphonuclear neutrophil recruitment in reperfused ischemic rabbit heart.

    PubMed Central

    Montrucchio, G.; Alloatti, G.; Mariano, F.; Comino, A.; Cacace, G.; Polloni, R.; De Filippi, P. G.; Emanuelli, G.; Camussi, G.

    1993-01-01

    This study investigated the role of platelet-activating factor in the recruitment of polymorphonuclear neutrophils (PMN) in a rabbit model of cardiac ischemia and reperfusion. The accumulation of PMN was evaluated 2 and 24 hours after removal of 40 minutes of coronary occlusion by morphometric analysis and 111In-labeled PMN infiltration. The administration of two structurally unrelated platelet-activating factor-receptor antagonists (SDZ 63-675, 5 mg/kg body weight, and WEB 2170, 5 mg/kg body weight) before reperfusion significantly reduced the accumulation of PMN, as well as the hemodynamic alterations and the size of necrotic area. Two hours after reperfusion, the percentage of increase of 111In-labeled PMN in transmural central ischemic zone was significantly reduced in rabbits pretreated with SDZ 63-675 (51.4 +/- 7.9) or WEB 2170 (32.4 +/- 8.8) with respect to untreated rabbits (107.6 +/- 13.5). The morphometric analysis of myocardial sections confirmed the reduction of PMN infiltration at 2 hours and demonstrated that at 24 hours the phenomenon was even more significant. In addition, SDZ 63-675 and WEB 2170 prevented early transient bradycardia and hypotension and reduced the infarct size, judged by staining with tetrazolium at 2 and 24 hours after reperfusion, and by histological examination at 24 hours. These results suggest that platelet-activating factor is involved in the accumulation of PMN in the reperfused ischemic myocardium and contributes to the evolution of myocardial injury. Images Figure 5 Figure 6 PMID:8434642

  6. 90Y-Labeled Anti-ROBO1 Monoclonal Antibody Exhibits Antitumor Activity against Small Cell Lung Cancer Xenografts

    PubMed Central

    Fujiwara, Kentaro; Koyama, Keitaro; Suga, Kosuke; Ikemura, Masako; Saito, Yasutaka; Hino, Akihiro; Iwanari, Hiroko; Kusano-Arai, Osamu; Mitsui, Kenichi; Kasahara, Hiroyuki; Fukayama, Masashi; Kodama, Tatsuhiko; Hamakubo, Takao; Momose, Toshimitsu

    2015-01-01

    Introduction ROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that 90Y-labeled anti-ROBO1 monoclonal antibody (90Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models. Methods For the biodistribution study, 111In-labeled anti-ROBO1 monoclonal antibody (111In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with 90Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out. Results As a result of the biodistribution study, we observed tumor uptake of 111In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of 111In-labeled anti-ROBO1. In the RIT study, 90Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur. Conclusions These results suggest that RIT with 90Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC. PMID:26017283

  7. Preclinical evaluation of 111In-DTPA-INCA-X anti-Ku70/Ku80 monoclonal antibody in prostate cancer

    PubMed Central

    Evans-Axelsson, Susan; Vilhelmsson Timmermand, Oskar; Welinder, Charlotte; Borrebaeck, Carl AK; Strand, Sven-Erik; Tran, Thuy A; Jansson, Bo; Bjartell, Anders

    2014-01-01

    The aim of this investigation was to assess the Ku70/Ku80 complex as a potential target for antibody imaging of prostate cancer. We evaluated the in vivo and ex vivo tumor targeting and biodistribution of the 111In-labeled human internalizing antibody, INCA-X (111In-DTPA-INCA-X antibody), in NMRI-nude mice bearing human PC-3, PC-3M-Lu2 or DU145 xenografts. DTPA-conjugated, non-labeled antibody was pre-administered at different time-points followed by a single intravenous injection of 111In-DTPA-INCA-X. At 48, 72 and 96 h post-injection, tissues were harvested, and the antibody distribution was determined by measuring radioactivity. Preclinical SPECT/CT imaging of mice with and without the predose was performed at 48 hours post-injection of labeled DTPA-INCA-X. Biodistribution of the labeled antibody showed enriched activity in tumor, spleen and liver. Animals pre-administered with DTPA-INCA-X showed increased tumor uptake and blood content of 111In-DTPA-INCA-X with reduced splenic and liver uptake. The in vitro and in vivo data presented show that the 111In-labeled INCA-X antibody is internalized into prostate cancer cells and by pre-administering non-labeled DTPA-INCA-X, we were able to significantly reduce the off target binding and increase the 111In-DTPA-INCA-X mAb uptake in PC-3, PC-3M-Lu2 and DU145 xenografts. The results are encouraging and identifying the Ku70/Ku80 antigen as a target is worth further investigation for functional imaging of prostate cancer. PMID:24982817

  8. Sequential assessment of pulmonary epithelial diethylene triamine penta-acetate clearance and intrapulmonary transferrin accumulation during Escherichia coli peritonitis

    SciTech Connect

    Ishizaka, A.; Stephens, K.E.; Segall, G.M.; Hatherill, J.R.; McDougall, I.R.; Wu, Z.; Raffin, T.A. )

    1990-03-01

    The individual roles of pulmonary capillary endothelial and alveolar epithelial permeability in the pathogenesis of the adult respiratory distress syndrome (ARDS) are unclear. We developed a method for the sequential assessment of pulmonary macromolecule accumulation and small solute clearance in vivo using a gamma camera. We measured the exponential clearance coefficient of 111In-labeled diethylene triamine penta-acetate (111In-DTPA) to assess airway clearance of small solutes. We also calculated the exponential equilibration coefficient of 111In-labeled transferrin (111In-TF) to assess intrapulmonary accumulation of transferrin. We determined these parameters in guinea pigs with Escherichia coli peritonitis and compared them with a saline-treated control group, oleic-acid-treated groups, and a group treated with low molecular weight dextran Ringer solution. The pulmonary DTPA clearance and the intrapulmonary transferrin accumulation were significantly increased in the peritonitis group (29.4 +/- 8.2 x 10(-3) min-1, p less than 0.02, and 15.1 +/- 3.1 x 10(-3) min-1, p less than 0.02) when compared with the control group (3.1 +/- 0.8 x 10(-3) min-1 and 4.5 +/- 0.5 x 10(-3) min-1). These changes developed within 5.5 h of the initial insult. Neither increased extravascular lung water nor elevated pulmonary artery and left atrial pressures were detected in the peritonitis group. The low molecular weight dextran Ringer group did not show a significant increase in the pulmonary DTPA clearance and the intrapulmonary transferrin accumulation.

  9. Alpha-melanocyte-stimulating hormone peptide analogs labeled with technetium-99m and indium-111 for malignant melanoma targeting.

    PubMed

    Chen, JianQing; Cheng, Zhen; Miao, Yubin; Jurisson, Silvia S; Quinn, Thomas P

    2002-02-15

    Previous studies have shown that the compact structure of a rhenium-cyclized alpha--melanocyte-stimulating hormone peptide analog, [Cys3410,D-Phe7]alpha-MSH(3--13), or Re-CCMSH, significantly enhanced its in vivo tumor uptake and retention. In this study, the metal chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminus of Re-CCMSH in order to develop a melanoma-targeting peptide that could be labeled with a wider variety of imaging and therapeutic radionuclides. Biodistribution properties of indium-111 ((111)In)--labeled DOTA-Re-CCMSH were compared with the non-DOTA-containing technetium-99m ((99m)Tc)--CCMSH in murine melanoma--bearing C57 mice to determine the effects of DOTA on tumor uptake and whole-body clearance. The tumor targeting capacity and clearance kinetics of (111)In-DOTA-Re-CCMSH were also compared with other related cyclic and linear (111)In-labeled DOTA-alpha-MSH complexes. The in vivo distribution data showed that the conjugation of DOTA to Re-CCMSH did not reduce its initial tumor uptake kinetics but did enhance its tumor retention and renal clearance properties. The tumor uptake of (111)In-DOTA-Re-CCMSH was significantly higher than the other (111)In-DOTA--coupled cyclic or linear alpha-MSH analogs used in this study. Moreover, (111)In-DOTA-Re-CCMSH displayed lower radioactivity accumulation in normal tissues of interest than its non-Re-cyclized counterpart, (111)In-DOTA-CCMSH; the disulfide bond--cyclized (111)In-DOTA-CMSH; or the linear (111)In-DOTA-NDP. Peptide cyclization via rhenium coordination significantly enhanced the tumor targeting and renal clearance properties of DOTA-Re-CCMSH, making it an excellent candidate for melanoma radiodetection and radiotherapy. Copyright 2002 American Cancer Society.

  10. Effects of sphincter of Oddi motility on the formation of cholesterol gallstones

    PubMed Central

    Rong, Zhong-Hou; Chen, Hong-Yuan; Wang, Xin-Xing; Wang, Zhi-Yi; Xian, Guo-Zhe; Ma, Bang-Zhen; Qin, Cheng-Kun; Zhang, Zhen-Hai

    2016-01-01

    AIM: To investigate the mechanisms and effects of sphincter of Oddi (SO) motility on cholesterol gallbladder stone formation in guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups, the control group (n = 10) and the cholesterol gallstone group (n = 24), which was sequentially divided into four subgroups with six guinea pigs each according to time of sacrifice. The guinea pigs in the cholesterol gallstone group were fed a cholesterol lithogenic diet and sacrificed after 3, 6, 9, and 12 wk. SO manometry and recording of myoelectric activity were obtained by a multifunctional physiograph at each stage. Cholecystokinin-A receptor (CCKAR) expression levels in SO smooth muscle were detected by quantitative real-time PCR (qRT-PCR) and serum vasoactive intestinal peptide (VIP), gastrin, and cholecystokinin octapeptide (CCK-8) were detected by enzyme-linked immunosorbent assay at each stage in the process of cholesterol gallstone formation. RESULTS: The gallstone formation rate was 0%, 0%, 16.7%, and 83.3% in the 3, 6, 9, and 12 wk groups, respectively. The frequency of myoelectric activity in the 9 wk group, the amplitude of myoelectric activity in the 9 and 12 wk groups, and the amplitude and the frequency of SO in the 9 wk group were all significantly decreased compared to the control group. The SO basal pressure and common bile duct pressure increased markedly in the 12 wk group, and the CCKAR expression levels increased in the 6 and 12 wk groups compared to the control group. Serum VIP was elevated significantly in the 9 and 12 wk groups and gastrin decreased significantly in the 3 and 9 wk groups. There was no difference in serum CCK-8 between the groups. CONCLUSION: A cholesterol gallstone-causing diet can induce SO dysfunction. The increasing tension of the SO along with its decreasing activity may play an important role in cholesterol gallstone formation. Expression changes of CCKAR in SO smooth muscle and serum

  11. [Effects of different fibre diameter of polypyrrole after direct current stimulation on adhesion and proliferation of cells and bacteria].

    PubMed

    Yang, Ying-ting; Wei, Yan; Xia, Bin; Wang, Zhen-gao

    2016-02-18

    To study the effects of cells'and bacteria's adhesion and proliferation on different fiber diameters of polypyrrole coating with electricity. Titanium coated with polypyrrole was divided into no electrical stimulation and stimulation groups, each group had 30-60 nm, 70- 100 nm, 130-170 nm diameters of the fiber. MC3T3 cells and Staphylococcus aureus (S. aureus) were inoculated on different fiber diameters of polypyrrole coating with and without electric stimulation. We gave the electrical stimulation group 100 mV for 1 h and every 24 hours gave it 1 h stimulation, and no electrical stimulation group was not managed. We used scanning electron microscope (SEM) to observe the cells'and bacteria's morphology. The cells were given 20 mL CCK-8 solutions after 1,3,7 days' cultivation, then incubated for 2 h, the solution was transferred to 96-well plate, we measured the cells' CCK-8 of the 30-60 nm, 70-100 nm, and 130-170 nm groups by Elisa. The cells on different fiber diameters were also stained by live-dead cell staining kit, TritonX-100 and DAPI. We used PBS to wash and glycerin to seal them. The live-dead situation and morphology were tested by co focal microscope. The bacterial were stained by Live/dead baclight bacterial viability kits, we detect the suspension's D of the 30-60 nm, 70-100 nm, and 130-170 nm groups, and also observed the bacteria's survival situation by co focal microscope. The CCK-8 of the cells with direct current stimulation was higher than that of the unpowered group (F=12.248, P=0.006). The smaller the fiber diameter, the better was the cell's adhesion and proliferation (F=9.261, P=0.005). The bacterial suspension's D of the electric group was lower than that of the unpowered group, and the fiber diameter had no significant effect on the bacteria's growth(F=9.641, P=0.036). Polypyrrole coating with electricity can promote the cell's proliferation and inhibit the bacteria's proliferation, and the cell growth on small fiber diameter coating is

  12. Effect of radiation on cytotoxicity, apoptosis and cell cycle arrest of human osteosarcoma MG-63 induced by a ruthenium(II) complex

    NASA Astrophysics Data System (ADS)

    Liu, Si-Hong; Zhao, Jian-Hua; Deng, Kun-Kang; Wu, Yong; Zhu, Jian-Wei; Liu, Qing-Hua; Xu, Hui-Hua; Wu, Hai-Feng; Li, Xin-Yan; Wang, Jian-Wei; Guo, Qi-Feng

    2015-04-01

    Radiation has large influence on the cytotoxicity, apoptosis and cell cycle arrest. The bioactivity of ruthenium(II) complex [Ru(dmb)2(DBHIP)](ClO4)2 (Ru1) (DBHIP = 2-(3,5-dibromo-4-hydroxylphenyl)imidazo[4,5-f][1,10]phenanthroline) was investigated in the absence and presence of radiation. The cytotoxicity of Ru1 against MG-63 cells was evaluated by CCK-8 method. Ru1 shows high cytotoxicity upon radiation. Radiation can enhance the cytotoxicity of Ru1 on MG-63 cells. The apoptosis was studied by Hoechst 33258 staining method and flow cytometry. The reactive oxygen species, mitochondrial membrane potential, cell cycle arrest and western blot analysis were investigated in detail. The complex induces the apoptosis in MG-63 cells through ROS-mediated mitochondrial dysfunction pathway.

  13. Synthesis of a Cationic BODIPY-Containing Conjugated Polymer for Detection of DNA and Cellular Imaging.

    PubMed

    Wang, Lingyun; Fang, Guipo; Cao, Derong

    2016-03-01

    A water-soluble cationic conjugated polyelectrolyte (P1) containing fluorene, BODIPY and diacetylene moieties was synthesized and characterized. P1 showed two main absorption bands with maxima at 360 and 574 nm as well as fluorescence maxima at 648 nm due to the incorporation of BODIPY into the polymer backbone. Addition of CT DNA can quench the emission of P1 because of the formation of a P1/CT DNA complex, which was demonstrated by UV-vis spectra and dynamic light scattering (DLS) analyses. Cellular imaging results indicated P1 could be utilized as cellular imaging of HeLa cells, where red fluorescence was observed in the partial cytoplasm. Moreover, CCK-8 assay showed P1 had a low cytotoxicity.

  14. Bax is not involved in the resveratrol-induced apoptosis in human lung adenocarcinoma cells

    NASA Astrophysics Data System (ADS)

    Zhang, Wei-wei; Wang, Zhi-ping; Chen, Tong-sheng

    2010-02-01

    Resveratrol (RV) is a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts. Previous studies indicate that RV has an ability to inhibit various stages of carcinogenesis and eliminate preneoplastic cells in vitro and in vivo. However, little is known about the molecular mechanism of RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cell. In this report, we analyzed whether Bax translocation from cytoplasm to mitochondria during RV-induced apoptosis in single living cell using onfocal microscopey. Cells were transfected with GFP-Bax plasmid. Cell counting kit (CCK-8) assay was used to assess the inhibition of RV on the cells viability. Apoptotic activity of RV was detected by Hoechst 33258 and propidium iodide (PI) staining. Our results showed that RV induced a dose-dependent apoptosis in which Bax did not translocate to mitochondrias.

  15. Self-assembly of amphiphilic tripeptides into nanoparticles for drug delivery.

    PubMed

    Tu, Zhaoxu; Xu, Xianghui; Jian, Yeting; Zhong, Dan; He, Bin; Gu, Zhongwei

    2014-01-01

    Peptide-based nanomaterials are widely used as nanocarriers for catalysis, drug delivery, and gene delivery. In this paper, we designed and synthesized the amphiphilic tripeptides through solution phase synthesis. The tripeptides were purified by column chromatography and the molecular structures were confirmed by (1)H NMR and TOF-MS. The tripeptides could self-assemble into spherical nanoparticles in aqueous media with a low critical aggregation concentration. The size and morphology of the nanoparticles were performed by dynamic light scattering, scanning electron microscopy and transmission electron microscope. The peptide-based nanoparticles were used as biocompatible nanocarriers for encapsulating hydrophobic doxorubicin (DOX) to achieve controlled release. The CCK-8 assay indicated that the peptide-based nanocarriers could enhance cellular uptake and drug efficacy of DOX to A549 tumor cell line. These results showed that the self-assembly of amphiphilic tripeptides provided a facile strategy to fabricate nanoparticles for anti-tumor drug delivery.

  16. Effect of radiation on cytotoxicity, apoptosis and cell cycle arrest of human osteosarcoma MG-63 induced by a ruthenium(II) complex.

    PubMed

    Liu, Si-Hong; Zhao, Jian-Hua; Deng, Kun-Kang; Wu, Yong; Zhu, Jian-Wei; Liu, Qing-Hua; Xu, Hui-Hua; Wu, Hai-Feng; Li, Xin-Yan; Wang, Jian-Wei; Guo, Qi-Feng

    2015-04-05

    Radiation has large influence on the cytotoxicity, apoptosis and cell cycle arrest. The bioactivity of ruthenium(II) complex [Ru(dmb)2(DBHIP)](ClO4)2 (Ru1) (DBHIP=2-(3,5-dibromo-4-hydroxylphenyl)imidazo[4,5-f][1,10]phenanthroline) was investigated in the absence and presence of radiation. The cytotoxicity of Ru1 against MG-63 cells was evaluated by CCK-8 method. Ru1 shows high cytotoxicity upon radiation. Radiation can enhance the cytotoxicity of Ru1 on MG-63 cells. The apoptosis was studied by Hoechst 33258 staining method and flow cytometry. The reactive oxygen species, mitochondrial membrane potential, cell cycle arrest and western blot analysis were investigated in detail. The complex induces the apoptosis in MG-63 cells through ROS-mediated mitochondrial dysfunction pathway.

  17. Chemical constituents of Narcissus tazetta var. chinensis and their antioxidant activities.

    PubMed

    Fu, Ka-Li; Li, Xian; Ye, Ji; Lu, Lu; Xu, Xi-Ke; Li, Hui-Liang; Zhang, Wei-Dong; Shen, Yun-Heng

    2016-09-01

    Two new flavan derivatives tazettones C-D (1-2), one new β-coumaranone (tazettone E, 3), one new flavan (tazettone F, 4), and one new phenylpropanoid (tazettone G, 5), together with six known flavonoids (6-11), were isolated from the bulbs of Narcissus tazetta var. chinensis Roem. Their structures were elucidated by spectroscopic analysis. In addition, the structures of 1-3 were confirmed by single crystal X-ray diffraction. All isolated compounds were tested for antioxidant activity by Cell Counting Kit-8 (CCK-8) assay. Compounds 6-8 and 10-11 exhibited potent antioxidant activity against H2O2-induced impairment in human SH-SY5Y neuroblastoma cells at tested concentrations. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Biocompatibility of nano-hydroxyapatite/polyetheretherketone composite materials with osteoblasts cultured in vitro

    NASA Astrophysics Data System (ADS)

    Wang, Lin; Huang, Feijuan; Wu, Zhengzhi; Ma, Rui

    2017-04-01

    The biocompatibility of the Sprague Dawley (SD) rat osteoblasts, which were cultured on the surfaces of nano-hydroxyapatite/polyetheretherketone (n-HA/PEEK) composites were investigated in this work. The osteoblasts of 24- hour old SD rats were cultured and identified by modified enzymatic digestion in vitro. The morphology and proliferation of cells were observed in CCK-8 regent staining, inverted microscopes, and by scanning electron microscopy (SEM) respectively. The results show that n-HA/PEEK composites have good biocompatibility with SD osteoblasts and that they can promote the growth of the cells that were cultured on the surfaces of the composites. The content of HA in n- HA/PEEK composites plays an important role in cell proliferation.

  19. Fluorescence imaging analysis of taxol-induced ASTC-a-1 cell death with cell swelling and cytoplasmic vacuolization

    NASA Astrophysics Data System (ADS)

    Chen, Tong-sheng; Sun, Lei; Wang, Longxiang; Wang, Huiying

    2008-02-01

    Taxol (Paclitaxel), an isolated component from the bark of the Pacific yew Taxus brevifolia, exhibits a broad spectrum of clinical activity against human cancers. Taxol can promote microtubule (MT) assembly, inhibit depolymerization, and change MT dynamics, resulting in disruption of the normal reorganization of the microtubule network required for mitosis and cell proliferation. However, the molecular mechanism of taxol-induced cell death is still unclear. In this report, CCK-8 was used to assay the inhibition of taxol on the human lung adenocarcinoma (ASTC-a-1) cells viability, confocal fluorescence microscope was used to monitor the morphology changes of cells with taxol treatment. We for the first time describe the characteristics of taxol-induced cells swelling, cytoplasmic vacuolization and cell death. Taxol induced swelling, cytoplasmatic vacuolization and cell death without cell shrinkage and membrane rupture. These features differ from those of apoptosis and resemble the paraptosis, a novel nonapoptotic PCD.

  20. Direct Solvent-Derived Polymer-Coated Nitrogen-Doped Carbon Nanodots with High Water Solubility for Targeted Fluorescence Imaging of Glioma.

    PubMed

    Wang, Yi; Meng, Ying; Wang, Shanshan; Li, Chengyi; Shi, Wei; Chen, Jian; Wang, Jianxin; Huang, Rongqin

    2015-08-05

    Cancer imaging requires biocompatible and bright contrast-agents with selective and high accumulation in the tumor region but low uptake in normal tissues. Herein, 1-methyl-2-pyrrolidinone (NMP)-derived polymer-coated nitrogen-doped carbon nanodots (pN-CNDs) with a particle size in the range of 5-15 nm are prepared by a facile direct solvothermal reaction. The as-prepared pN-CNDs exhibit stable and adjustable fluorescence and excellent water solubility. Results of a cell viability test (CCK-8) and histology analysis both demonstrate that the pN-CNDs have no obvious cytotoxicity. Most importantly, the pN-CNDs can expediently enter glioma cells in vitro and also mediate glioma fluorescence imaging in vivo with good contrast via elevated passive targeting. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Induction of Apoptosis in Human Leukemic Cell Lines by Diallyl Disulfide via Modulation of EGFR/ERK/PKM2 Signaling Pathways.

    PubMed

    Luo, Nian; Zhao, Lv-Cui; Shi, Qing-Qiang; Feng, Zi-Qiang; Chen, Di-Long; Li, Jing

    2015-01-01

    Diallyl disulfide (DADS) may exert potent anticancer action both in vitro and in vivo. Although its effects on cancer are significant, the underlying mechanisms remain unknown. In this study, we sought to elucidate possible links between DADS and pyruvate kinase (PKM2). KG1α, a leukemia cell line highly expressing PKM2 was used with a cell counting kit (CCK)-8 and flow cytometry (FCM) to investigate the effects of DADS. Relationships between PKM2 and DADS associated with phosphorylation of EGFR, ERK1/2 and MEK, were assessed by western blot analysis. In KG1α cells highly expressing PKM2, we found that DADS could affect proliferation, apoptosis and EGFR/ERK/PKM2 signaling pathways, abrogating EGF-induced nuclear accumulation of PKM2. These results suggested that DADS suppressed the proliferation of KG1α cells, providing evidence that its proapoptotic effects are mediated through the inhibition of EGFR/ERK/PKM2 signaling pathways.

  2. Spy1 participates in the proliferation and apoptosis of epithelial ovarian cancer.

    PubMed

    Lu, Shumin; Liu, Rong; Su, Min; Wei, Yingze; Yang, Shuyun; He, Song; Wang, Xia; Qiang, Fulin; Chen, Chen; Zhao, Shuyang; Zhang, Weiwei; Xu, Pan; Mao, Guoxin

    2016-02-01

    This study focused on determining the role of Spy1 in human epithelial ovarian cancer (EOC). Speedy is a novel cell cycle protein capable of promoting cell proliferation. In this study, western blot and immunohistochemistrical analyses were performed to detect the expression of Spy1 in ovarian cancer. Spy1 protein levels increased with ovarian cancer grade, and Kaplan-Meier curve showed that overexpression of Spy1 was significantly correlated with reduced patient survival. In vitro, Spy1 depletion in ovarian cell lines led to reduced proliferation according to CCK8 and plate colony assays. The expression of Spy1 was positively related to pThr187-p27. Flow cytometry revealed that the reduced expression of Spy1 induced the apoptosis of the EOC cells. In summary, our findings suggested that Spy1 may be a novel independent prognostic predictor of survival for ovarian patients.

  3. Unexpected relationship between plasma protein binding and the pharmacodynamics of 2-NAP, a CCK1-receptor antagonist

    PubMed Central

    Gerskowitch, V P; Hodge, J; Hull, R A D; Shankley, N P; Kalindjian, S B; McEwen, J; Black, J W

    2007-01-01

    What is already known about this subject? Two chemically diverse CCK1 receptor antagonists have been shown clinically to inhibit CCK-evoked contraction of human gallbladder [2, 3]. These studies have not examined the relationship between plasma concentration and effect, the latter usually considered to be predictive from the free drug concentration [8]. We wanted to examine our novel CCK1 receptor antagonist in this validated model and also to explore its PK-PD relationship. What this study adds 2-NAP inhibited CCK-evoked human gallbladder contraction in vivo but at a plasma free concentration that was, in theory, too low to have achieved adequate CCK1 receptor occupancy. The study serves as a caveat to the assumption that free plasma concentration can be used to predict pharmacological effect. Aims To study the pharmacokinetics and pharmacodynamics of 2-NAP (2-naphthalenesulfonyl-L-aspartyl-(2-phenethyl)amide), a selective CCK1 receptor antagonist in healthy volunteers. Methods 2-NAP was given to 12 healthy male volunteers in an ascending dose, safety and PK phase 1a study by 1 h i.v. infusion (0.6–9.6 mg kg−1 h−1). A further 12 healthy male volunteers received i.v. CCK-8S (6.25 pmol kg−1 h−1) to produce gallbladder contraction, measured by ultrasound recordings of gallbladder volume, and the effect of concurrent i.v. 2-NAP administration was studied. Plasma protein binding in vitro and ex vivo was measured by ultrafiltration and by equilibrium dialysis. Results 2-NAP was generally well tolerated, displayed linear pharmacokinetics and a very high degree of plasma protein binding (99.9%). A 105 min i.v. CCK-8S infusion induced a reduction in gallbladder volume of 14.9 (±7.0) ml during placebo co-infusion and this was reduced to 2.4 (±5.9) ml when 2-NAP was co-infused with CCK-8S (P = 0.00024, paired t-test, mean change 12.5 ml; 95% CI For mean 7.4, 18.3 ml). This extent of inhibition was consistent with a 2-NAP total plasma concentration of 36 µm, but

  4. USP21 promotes cell proliferation and metastasis through suppressing EZH2 ubiquitination in bladder carcinoma

    PubMed Central

    Chen, Yong; Zhou, Bo; Chen, Daihui

    2017-01-01

    Bladder cancer (BC) is the second most common malignant tumor of the urinary tract in the world. In this study, we found that ubiquitin-specific protease (USP21) was upregulated in BC and the ectopic expression of USP21 was closely associated with tumor size and metastasis. Moreover, patients with higher levels of USP21 had poorer survival rate. Multiple function analysis such as CCK-8, colony formation, wound healing, and transwell analysis indicated that USP21 regulated cell proliferation and metastasis in bladder carcinoma cell lines. We also found that USP21 could facilitate epithelial–mesenchymal transition. As EZH2 has been reported to promote cell metastasis in BC, our work identified that USP21 deubiquitinated EZH2 and stabilized it. Our data demonstrated that USP21 might play a crucial role in regulating BC progression and could provide a potential therapeutic strategy for BC. PMID:28223825

  5. [Experiment studies on growth and proliferation of Hep-2 cells by silence of TRF2 gene].

    PubMed

    Jiao, Luyan; Xu, Yong; Tao, Zezhang

    2010-12-01

    To investigate the effect of RNA interference silencing telomere repeat factor 2 by observing Hep-2 cells' proliferation and apoptosis in larynx carcinoma cell line. A recombinant plasmid containing a single shRNA (shTRF2) was constructed. The expression of TRF2 gene was detected by PCR and cell proliferation was examined using CCK-8. Hep-2 cells' apoptosis was detected by flow cytometer (FCM). After treatment with shTRF2, the expression of TRF2 was distinctively depressed, and Hep-2 cells proliferation was obviously inhibited. Compared with control and negative group, cells with treatment of RNAi exhibited significantly more apoptosis. Using RNA interference technique to silence TRF2 gene is effective on inhibiting cancer cells' proliferation and help to induce cancer cells' apoptosis.

  6. Overexpression of Dishevelled-2 contributes to proliferation and migration of human esophageal squamous cell carcinoma.

    PubMed

    Zhou, Guoren; Ye, Jinjun; Sun, Lei; Zhang, Zhi; Feng, Jifeng

    2016-06-01

    Dishevelled-2 (Dvl2) was associated with tumor cell proliferation and migration. We aimed to examine the mechanism of Dvl2 in esophageal squamous cell carcinoma (ESCC). Dvl2 was overexpressed in human ESCC tissues and cell lines ECA109 and TE1 cells. CCK-8 and colony formation assay was performed to evaluate the proliferation in ECA109 cells transfected with Dvl2-shRNA. Wound-healing assay and transwell assay were used to examine the activities of migration and invasion in Dvl2-silenced ESCC cells. Knockdown of Dvl2 significantly reduced ECA109 cell proliferation and migration. Moreover, we demonstrated that the proliferation and migration ability of Dvl2 might through the activation of Wnt pathway by targeting the Cyclin D1 and MMP-9. We came to the conclusion that the proliferation and migration effects of Dvl2 might contribute to malignant development of human ESCC.

  7. Analysis on the Alpinia katsumadai components of Zingiberaceae plants and their functions on myeloma resistance.

    PubMed

    Wang, Jue; Qiu, Rubiao; Yuan, Lianjing; Meng, Fei; Tang, Qian

    2015-05-01

    Generally speaking, zingiberaceae plants with sweet fragrance are commonly seen as perennial herbs that contains numerous well-known crude drugs and fragrant plants like Amomum villosum, Amomumtsao-ko, Ginger, Alpinia katsumadai and Radix curcumae, which are widely used in daily life. This paper analyzed chemical components of Alpinia katsumadai of zingiberaceae and applied several laminar analysis to further develop its active ingredients, aiming to make sure its function on tumor assistance. Actually, cardamomin contained in Alpinia katsumadai has been recorded to act notably in myeloma resistance, which was verified by cholecystokinin-octopeptide (CCK-8) in this paper. Cardamom in is proved to have multiple anti-myeloma effects, including myeloma cell activity and proliferation control, cell cycle retardant and apoptosis induction, which indicates its value in the field of medical pharmacy.

  8. Exploration for the multi-effect of cardamom in's resistance to multiple myeloma.

    PubMed

    Zhihua, Zhao; Jianping, Yang; Miaomiao, Sun; Kuisheng, Chen

    2014-11-01

    This paper aimed to probe the cardamom in effect on the viability, proliferation, apoptosis and periodic function of the multiple myeloma, and explore its mechanism. We used CCK-8 method to evaluate the effect of cardamom in on the viability of PBMNCs (Persom Blood Mononuclear Normal Cells). EdU can test the influence of small cell proliferation. We used the method of PI single-staining flow cytometry, in order to test the influence of tumor cell cycle. AO (Acridine Orange), EB (Ethidium Bromide) double staining fluorescene microscope was applied to observe the influence of tumor apoptotic morphology. It can be concluded that cardamom in can inhibit the viability and proliferation of MM (Multiple Myeloma) cells and cardamom in is the anti-myeloma drug with strong viability.

  9. New Alkaloids from Green Vegetable Soybeans and Their Inhibitory Activities on the Proliferation of Concanavalin A-Activated Lymphocytes.

    PubMed

    Wang, Taoyun; Zhao, Jianping; Li, Xiaoran; Xu, Qiongming; Liu, Yanli; Khan, Ikhlas A; Yang, Shilin

    2016-03-02

    A comprehensive phytochemical study of the chemical constituents of green vegetable soybeans resulted in the isolation of two new alkaloids, soyalkaloid A, 1, and isoginsenine, 2, together with four known ones, ginsenine, 3, (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 4, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 5, and indole-3-carboxylic acid, 6. The structures of compounds 1-6 were elucidated on the basis of spectroscopic and chemical analyses. All of the alkaloids were isolated from soybeans for the first time, and compound 1 was a new indole-type alkaloid with a novel carbocyclic skeleton. Their inhibitory activities on the proliferation of concanalin A-activated lymphocytes were assessed by CCK8 assay.

  10. Bax translocation into mitochondria during dihydroartemisinin(DHA)-induced apoptosis in human lung adenocarcinoma cells

    NASA Astrophysics Data System (ADS)

    Lu, Ying-ying; Chen, Tong-sheng; Qu, Jun-Le

    2009-02-01

    Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. This study was investigated in human lung adenocarconoma ASTC-a-1 cell line and aimed to determine whether the apoptotic process was mediated by Bax activation and translocation during DHA-induced apoptosis. In this study, DHA induced a time-dependent apoptotic cell death, which was assayed by Cell Counting Kit (CCK-8) and Hoechst 33258 staining. Detection of Bax aggregation and translocation to mitochondria was observed in living cells which were co-transfected with GFP-Bax and Dsred-mito plasmid using confocal fluorescence microscope technique. Overall, these results demonstrated that Bax activation and translocation to mitochondria occurred during DHA-induced apoptosis.

  11. Cholecystokinin-gated currents in neurons of the rat solitary complex in vitro.

    PubMed

    Branchereau, P; Champagnat, J; Denavit-Saubié, M

    1993-12-01

    1. Ionic conductances controlled by type A and type B cholecystokinin (CCK) receptors were studied in neurons of the rat nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNV), using intracellular and whole-cell patch clamp recordings in current or voltage clamp configuration during bath application of agonists (CCK8, CCK4, BC 264) and antagonists. 2. CCKA receptor-related inhibition was associated with a membrane hyperpolarization and a decrease in input resistance that developed 2-6 min after the arrival of drug into the extracellular medium. These effects were induced by 5 nM CCK8 but not BC 264 and they were blocked by the CCKA antagonist, L-364,718, but not by the CCKB antagonist, L-365,260. 3. CCKA receptor-related inhibition was generated by a potassium current that reversed at a reversal potential E(rev) of -73 +/- 1 (mean +/- SE) mV with bathing potassium concentration [K+]o = 6 mM and at -88 +/- 1 with [K+]o = 3 mM, in agreement with the Nernst equation for potassium ions. 4. CCKB receptor-related excitation was associated with a membrane depolarization and an increase of the input resistance induced by the following agonists at threshold concentrations: CCK8 (0.2 nM) > or = BC 264 (0.4 nM) > CCK4 (10.9 nM). The increase of input resistance was abolished by L-365,260 and was maintained after blockade of the CCKA current by L-364,718. 5. CCKB receptor-related excitation, in the neurons (30% of cases) in which clear response reversal was observed, appeared to be generated by a decrease of a potassium conductance. Responses showed a reversal potential E(rev) of -68 +/- 4 mV with [K+]o = 6 mM and -89 +/- 1 mV with [K+]o = 3 mM, verifying predictions from the Nernst equation applied to potassium ions. However, in 70% of cases, clear reversal was not observed at membrane potentials negative to the theoretical potassium equilibrium potential EK. 6. In voltage clamp studies, CCK8 induced a 181 +/- 17 pA inward current associated with a 26

  12. Cholecystokinin-33 acutely attenuates food foraging, hoarding and intake in Siberian hamsters

    PubMed Central

    Teubner, Brett J.W.; Bartness, Timothy J.

    2009-01-01

    Neurochemicals that stimulate food foraging and hoarding in Siberian hamsters are becoming more apparent, but we do not know if cessation of these behaviors is due to waning of excitatory stimuli and/or the advent of inhibitory factors. Cholecystokinin (CCK) may be such an inhibitory factor as it is the prototypic gastrointestinal satiety peptide and is physiologically important in decreasing food intake in several species including Siberian hamsters. Systemic injection of CCK-33 in laboratory rats decreases food intake, doing so to a greater extent than CCK-8. We found minimal effects of CCK-8 on food foraging and hoarding previously in Siberian hamsters, but have not tested CCK-33. Therefore, we asked: Does CCK-33 decrease normal levels or food deprivation-induced increases in food foraging, hoarding and intake? Hamsters were housed in a wheel running-based foraging system with simulated burrows to test the effects of peripheral injections of CCK-33 (13.2, 26.4, or 52.8 μg/kg body mass), with or without a preceding 56 h food deprivation. The highest dose of CCK-33 caused large baseline reductions in all three behaviors for the 1st h post injection compared with saline; in addition, the intermediate CCK-33 dose was sufficient to curtail food intake and foraging during the 1st h. In food deprived hamsters, we used a 52.8 μg/kg body mass dose of CCK-33 which decreased food intake, hoarding, and foraging almost completely compared with saline controls for 1 h. Therefore, CCK-33 appears to be a potent inhibitor of food intake, hoarding, and foraging in Siberian hamsters. PMID:20025915

  13. Synthesis and biological activities of pseudopeptide analogues of the C-terminal heptapeptide of cholecystokinin. On the importance of the peptide bonds.

    PubMed

    Rodriguez, M; Lignon, M F; Galas, M C; Fulcrand, P; Mendre, C; Aumelas, A; Laur, J; Martinez, J

    1987-08-01

    A series of pseudopeptide analogues of the C-terminal heptapeptide of cholecystokinin in which each peptide bond, one at a time, has been replaced by a CH2NH bond were synthesized: Z-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp psi-(CH2NH)Phe-NH2 (1), Z-Tyr(SO3-)-Nle-Gly-Trp-Nle psi (CH2NH)Asp-Phe-NH2 (2), Z-Tyr(SO3-)-Nle-Gly-Trp psi-(CH2NH)Nle-Asp-Phe-NH2 (3), Z-Tyr(SO3-)-Nle-Gly psi(CH2NH)Trp-Nle-Asp-Phe-NH2 (4), Z-Tyr(SO3-)-Nle psi-(CH2NH)Gly-Trp-Nle-Asp-Phe-NH2 (5), Z-Tyr(SO3-)-Met-Gly-Trp-Nle-Asp psi (CH2NH)Phe-NH2 (6), Z-Tyr-(SO3-)-Met-Gly-Trp-Nle psi (CH2NH)Asp-Phe-NH2 (7), Z-Tyr(SO3-)-Met-Gly-Trp psi (CH2NH)Nle-Asp-Phe-NH2 (8). These derivatives were studied for their ability to stimulate amylase release from rat pancreatic acini and to inhibit the binding of labeled CCK-9 to rat pancreatic acini and to guinea pig brain membrane CCK receptors. They were compared to the potent CCK-8 analogue Boc-Asp-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2. All of these pseudopeptides were able to stimulate amylase secretion with the same efficacy as CCK-8 but with varying potencies. These compounds were also potent in inhibiting the binding of labeled CCK-9 to CCK receptors from rat pancreatic acini and from guinea pig brain membranes.

  14. Systemic administration of anorexic gut peptide hormones impairs hedonic-driven sucrose consumption in mice.

    PubMed

    Yamaguchi, Erina; Yasoshima, Yasunobu; Shimura, Tsuyoshi

    2017-03-15

    A number of reports suggest that gut hormones such as cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and peptide YY(3-36) (PYY3-36), which are released postprandially, suppress homeostatic food intake and result in satiety and the termination of feeding. However, it remains unclear whether these peptide hormones also suppress non-homeostatic consumption of palatable foods or fluids. To examine whether gut hormones reduce hedonically motivated sugar consumption, we assessed the effects of intraperitoneal administration of these gut hormones on the consumption of a highly palatable sucrose solution, using a mouse model we previously established for binge-like sucrose overconsumption (Yasoshima and Shimura, 2015). To reduce homeostatic hunger, chow was available at nighttime prior to testing. After a limited-access training procedure for 10days, during which access to both sucrose and chow were controlled, on the test day, control mice injected with saline consumed significantly more sucrose than during the pre-training period. In contrast, sucrose consumption on the test day in the mice injected with CCK-8 (2 and 4μg/kg), GLP-1 (500 and 1000nmol/kg), or PYY3-36 (12.5 and 25nmol/kg) was significantly less than that in saline-injected mice. In a separate cohort of mice, the higher doses of CCK-8 and GLP-1 and a greater dose of PYY3-36 (50nmol/kg) did not produce conditioned taste aversion to saccharin, suggesting that the doses of exogenous hormones in the present study do not cause aversive visceral distress. The present findings suggest that the systemic administration of these three gut hormones suppresses hedonic-driven sugar consumption due to the anorexic, but not aversive-visceral, effects of these hormones. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Histone demethylase JMJD1C regulates esophageal cancer proliferation Via YAP1 signaling

    PubMed Central

    Cai, Yixin; Fu, Xiangning; Deng, Yu

    2017-01-01

    Esophageal cancer (EC) is the most lethal cancer, and it is of significant concern worldwide, particularly in China. However, there are no effective treatments to cure it, such as chemotherapy, surgery, or radiotherapy. This is attributed to the lack of understanding of the molecular mechanisms of EC. Recently, the superfamily of Jmj-containing KDMs has been shown to play an important role in tumorigenesis in various cancers, including EC. In this study, we demonstrated that JMJD1C was upregulated in patient EC tissues and different EC cell lines. Furthermore, JMJD1C levels were positively correlated with the TNM stage. Moreover, the colony formation assay, CCK8, and cell number count assay showed that the knockdown of JMJD1C inhibited EC cell proliferation. Western blot analysis and the quantitative real-time polymerase chain reaction assay showed that the knockdown of JMJD1C repressed the protein and mRNA levels of YAP1 via regulating the H3K9me2 activity, but not the H3K9me1 activity. The colony formation assay, CCK8 analysis, and cell number count assay revealed that inhibition of EC cell proliferation by the knockdown of JMJD1C was rescued by overexpression of YAP1. Taken together, our results demonstrated that JMJD1C controls the proliferation of EC via modulation of H3K9me2 activity, targeting the YAP1 gene expression and functions as a tumor suppressor in EC. This novel pathway may serve as a therapeutic target for EC patients. PMID:28123852

  16. Vagal afferent-dependent cholecystokinin modulation of visceral pain requires central amygdala NMDA-NR2B receptors in rats.

    PubMed

    Wang, E M; Li, W T; Yan, X J; Chen, X; Liu, Q; Feng, C C; Cao, Z J; Fang, J Y; Chen, S L

    2015-09-01

    Cholecystokinin (CCK), a gut hormone that is released during feeding, exerts gastrointestinal effects in part through vagal pathway. It is reported to be a potential trigger for increased postprandial visceral sensitivity in healthy subjects and, especially in patients with irritable bowel syndrome. NR2B-containing N-methyl-d-aspartate (NMDA) receptors in the central amygdala (CeA) participate in pain modulation. Systemically administered CCK activates the CeA-innervating neurons. Here, we investigated whether CCK modulation of visceral sensitivity is mediated through CeA NMDA-NR2B receptors and whether this modulation involves vagal pathway. We first examined the visceromotor response (VMR) to colorectal distention (CRD) following i.p. injection of CCK octapeptide (CCK-8) in a rat model. Next, the NR2B antagonist ifenprodil and the NR2A antagonist NVP-AAM077 were microinjected into the CeA before systemic CCK injection. NR2B phosphorylation was detected by Western blot. To down-regulate NR2B gene expression, NR2B-specific small interfering RNA (siRNA) was delivered into CeA neurons by electroporation. In addition, the effects of functional deafferentation by perivagal application of capsaicin and pretreatment with the CCK1 receptor antagonist devazepide were investigated. CCK-8 increased VMR to CRD in a dose-dependent manner. This effect was blunted by intra-CeA administration of ifenprodil (but not NVP-AAM077) and was accompanied by phosphorylation of NR2B subunits in the CeA. CCK failed to increase VMR to CRD in NR2B siRNA-treated rats. Perivagal capsaicin application and pretreatment with devazepide prevented CCK-induced pronociception and CeA NR2B phosphorylation. The pronociception induced by systemic CCK, which is vagal afferent-dependent, requires activation of CeA NMDA-NR2B receptors. © 2015 John Wiley & Sons Ltd.

  17. [Evaluation of biocompatibility of Ti-6Al-4V scaffolds fabricated by electron beam melting].

    PubMed

    Wang, H; Zhao, B J; Yan, R Z; Wang, C; Luo, C C; Hu, M

    2016-11-09

    Objective: To investigate the biocompatibility of Ti-6Al-4V scaffolds fabricated by electron beam melting(EBM). Methods: Bone marrow mesenchymal stem cells(BMSC) co-cultured with Ti-6Al-4V specimens fabricated with EBM was prepared as experimental group and the regular cells culture was employed as control. The biocompatibility was detected using CCK-8 and cytoskeleton staining. The osteogenic differentiation ability was assessed using mineralization nodule formation. A 24 mm defect was created on the right mandibular body in 12 beagles. The mandibular defects were repaired with Ti-6Al-4V scaffolds mesh fabricated by EBM. General observation, CT and histology examination was carried out to evaluated the biocompatibility of Ti-6Al-4V scaffolds in vivo. Results: CCK-8 result showed the A values of the two groups had no significant difference(P >0.05). There was no significant difference between the two groups (P>0.05). Cytoskeletal staining showed that cells were fully stretched out and grew well on T-i6Al-4V specimen. The actin fibers were arranged in parallel and stained uniformly with fluorescent. After osteogenic culture, the quantity of the nodule formation of the experimental group and control group were 5.7±0.7 and 5.1 ± 0.6, respectively(P>0.05). All animals had tolerated the surgery and healed well. CT examination showed that Ti-6Al-4V scaffolds mesh had good retention with surrounding bone and the continuity of mandible was restored. Histological examination showed that no inflammation reaction or toxity was caused in the soft tissue surrounding the scaffolds and in the liver and kidney after implantation. Ti-6Al-4V scaffolds had good retention with surrounding bone. Conclusions: Ti-6Al-4V fabricated with electron beam melting has good biocompatibility.

  18. Proliferation and odontogenic differentiation of BMP2 gene-transfected stem cells from human tooth apical papilla: An in vitro study

    PubMed Central

    ZHANG, WEN; ZHANG, XIAOLEI; LING, JUNQI; LIU, WEI; ZHANG, XINCHUN; MA, JINGLEI; ZHENG, JIANMAO

    2014-01-01

    Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue-derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral-mediated BMP2 gene-transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector-transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK-8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: i) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ii) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; iii) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro. PMID:25070743

  19. Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy.

    PubMed

    Xu, Wei-li; Li, Suo-lin; Wen, Ming; Wen, Jun-ye; Han, Jie; Zhang, Hong-zhen; Gao, Fei; Cai, Jian-hui

    2013-08-01

    Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers, immunophenotype, tumor-specificity, and in vivo residence time, migration, and distribution. Therefore, tracing in vivo persistence, migration, and distribution of CTLs is important for cancer immunotherapy. Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared. Additionally, CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma, and their residence time, migration, and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values. Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed. Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity. No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P = 0.849) or interleukin-2 (P = 0.318) and interferon-γ (P = 0.201) levels. Distribution of CTLs in vivo varied with time. A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed. CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion. CTLs were observed up to three weeks later in the tumor, liver, kidneys, and spleen; this was related to the abundant blood supply or the nature of immune organs. CCK-8 assay is a novel method to select optimal CFSE staining concentrations. Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors. CFSE fluorescent markers can trace in vivo CTL persistence, migration, and distribution because of its stability

  20. Regulation of feeding behavior and food intake by appetite-regulating peptides in wild-type and growth hormone-transgenic coho salmon.

    PubMed

    White, Samantha L; Volkoff, Helene; Devlin, Robert H

    2016-08-01

    Survival, competition, growth and reproductive success in fishes are highly dependent on food intake, food availability and feeding behavior and are all influenced by a complex set of metabolic and neuroendocrine mechanisms. Overexpression of growth hormone (GH) in transgenic fish can result in greatly enhanced growth rates, feed conversion, feeding motivation and food intake. The objectives of this study were to compare seasonal feeding behavior of non-transgenic wild-type (NT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch), and to examine the effects of intraperitoneal injections of the appetite-regulating peptides cholecystokinin (CCK-8), bombesin (BBS), glucagon-like peptide-1 (GLP-1), and alpha-melanocyte-stimulating hormone (α-MSH) on feeding behavior. T salmon fed consistently across all seasons, whereas NT dramatically reduced their food intake in winter, indicating the seasonal regulation of appetite can be altered by overexpression of GH in T fish. Intraperitoneal injections of CCK-8 and BBS caused a significant and rapid decrease in food intake for both genotypes. Treatment with either GLP-1 or α-MSH resulted in a significant suppression of food intake for NT but had no effect in T coho salmon. The differential response of T and NT fish to α-MSH is consistent with the melanocortin-4 receptor system being a significant pathway by which GH acts to stimulate appetite. Taken together, these results suggest that chronically increased levels of GH alter feeding regulatory pathways to different extents for individual peptides, and that altered feeding behavior in transgenic coho salmon may arise, in part, from changes in sensitivity to peripheral appetite-regulating signals.

  1. Metformin Inhibits TGF-β1-Induced Epithelial-to-Mesenchymal Transition via PKM2 Relative-mTOR/p70s6k Signaling Pathway in Cervical Carcinoma Cells.

    PubMed

    Cheng, Keyan; Hao, Min

    2016-11-30

    Epithelial-to-mesenchymal transition (EMT) plays a prominent role in tumorigenesis. Metformin exerts antitumorigenic effects in various cancers. This study investigated the mechanisms of metformin in TGF-β1-induced Epithelial-to-mesenchymal transition (EMT) in cervical carcinoma cells. cells were cultured with 10 ng/mL TGF-β1 to induce EMT and treated with or without metformin. Cell viability was evaluated by CCK-8 (Cell Counting Kit 8, CCK-8) assay; apoptosis were analyzed by flow cytometry; cell migration was evaluated by wound-healing assay. Western blotting was performed to detect E-cadherin, vimentin, signal transducer and activator of transcription 3 (STAT3), snail family transcriptional repressor 2 (SNAIL2), phosphorylation of p70s6k (p-p70s6k) and -Pyruvate kinase M2 (PKM2) Results: TGF-β1 promoted proliferation and migration, and it attenuated apoptosis compared with cells treated with metformin with or without TGF-β1 in cervical carcinoma cells. Moreover, metformin partially abolished TGF-β1-induced EMT cell proliferation and reversed TGF-β1-induced EMT. In addition, the anti-EMT effects of metformin could be partially in accord with rapamycin, a specific mTOR inhibitor. Metformin decreased the p-p70s6k expression and the blockade of mTOR/p70s6k signaling decreased PKM2 expression. Metformin abolishes TGF-β1-induced EMT in cervical carcinoma cells by inhibiting mTOR/p70s6k signaling to down-regulate PKM2 expression. Our study provides a novel mechanistic insight into the anti-tumor effects of metformin.

  2. Sam68 regulates cell proliferation and cell adhesion-mediated drug resistance (CAM-DR) via the AKT pathway in non-Hodgkin's lymphoma.

    PubMed

    Wu, Yaxun; Xu, Xiaohong; Miao, Xiaobing; Zhu, Xinghua; Yin, Haibing; He, Yunhua; Li, Chunsun; Liu, Yushan; Chen, Yali; Lu, Xiaoyun; Wang, Yuchan; He, Song

    2015-12-01

    Sam68 (Src-associated in mitosis 68 kDa), a substrate for tyrosine kinase c-Src during mitosis, is up-regulated in a variety of human cancers and acts oncogenically promoting tumour progression. This study has explored biological function and clinical significance of Sam68 in non-Hodgkin's lymphoma (NHL). To examine Sam68 expression in NHL, clinically, eight diffuse large B-cell lymphomas and four reactive lymphoid hyperplasia fresh-frozen tissues were obtained for western blot and quantitative real-time PCR analyses. Using immunohistochemical staining, paraffin wax embedded sections from 164 cases of NHL patients were used to evaluate prognostic value of Sam68. Cell Counting Kit-8 (CCK-8) and soft agar colony assays were conducted to investigate the role of Sam68 in cell viability and cell proliferation respectively. Furthermore, effects of Sam68 on cell adhesion-mediated drug resistance (CAM-DR) was determined by CCK-8 assay and flow cytometric analysis. Expression status of Sam68 inversely correlated with clinical outcomes of patients with NHL, and it was also an independent prognostic factor for the outcomes. In addition, Sam68 was associated with proliferation of NHL cells. Knock-down of its gene inhibited cell proliferation and colony formation by delaying cell cycle progression. Furthermore, OCI-Ly8 and Jeko-1 cells adhering to FN and HS-5 expressed higher Sam68 protein, compared to their suspension counterparts. Sam68 promoted cell adhesion-mediated drug resistance (CAM-DR) via the AKT pathway. Increased Sam68 expression in NHL resulted in poor prognosis, and it promoted CAM-DR in NHL via AKT. © 2015 John Wiley & Sons Ltd.

  3. Silver nanoparticles promote osteogenic differentiation of human urine-derived stem cells at noncytotoxic concentrations.

    PubMed

    Qin, Hui; Zhu, Chen; An, Zhiquan; Jiang, Yao; Zhao, Yaochao; Wang, Jiaxin; Liu, Xin; Hui, Bing; Zhang, Xianlong; Wang, Yang

    2014-01-01

    In tissue engineering, urine-derived stem cells are ideal seed cells and silver nanoparticles (AgNPs) are perfect antimicrobial agents. Due to a distinct lack of information on the effects of AgNPs on urine-derived stem cells, a study was conducted to evaluate the effects of silver ions and AgNPs upon the cytotoxicity and osteogenic differentiation of urine-derived stem cells. Initially, AgNPs or AgNO3 were exposed to urine-derived stem cells for 24 hours. Cytotoxicity was measured using the Cell Counting kit-8 (CCK-8) test. The effects of AgNPs or AgNO3 at the maximum safety concentration determined by the CCK-8 test on osteogenic differentiation of urine-derived stem cells were assessed by alkaline phosphatase activity, Alizarin Red S staining, and the quantitative reverse transcription polymerase chain reaction. Lastly, the effects of AgNPs or AgNO3 on "urine-derived stem cell actin cytoskeleton organization" and RhoA activity were assessed by rhodamine-phalloidin staining and Western blotting. Concentration-dependent toxicity was observed starting at an AgNO3 concentration of 2 μg/mL and at an AgNP concentration of 4 μg/mL. At these concentrations, AgNPs were observed to promote osteogenic differentiation of urine-derived stem cells, induce actin polymerization and increase cytoskeletal tension, and activate RhoA; AgNO3 had no such effects. In conclusion, AgNPs can promote osteogenic differentiation of urine-derived stem cells at a suitable concentration, independently of silver ions, and are suitable for incorporation into tissue-engineered scaffolds that utilize urine-derived stem cells as seed cells.

  4. A Comparative Study on the Biological Characteristics of Human Adipose-Derived Stem Cells from Lipectomy and Liposuction

    PubMed Central

    Li, Wangzhou; Lei, Zhanjun; Li, Yuejun; Li, Xueyong

    2016-01-01

    Purposes To compare the biological behaviors of human adipose-derived stem cells (ADSCs) isolated from adipose tissue by lipectomy and liposuction, with the purpose of providing the basis for clinical application. Methods The proliferation and apoptosis of ADSCs were analyzed by CCK-8 assay and flow cytometry. Cell migration was measured by a wound healing assay. An ELISA assay was used to evaluate paracrine functions. SOD and MDA were tested by xanthine oxidase and thiobarbituric acid reactions, respectively. In addition, we used a CCK-8, LDH assay and flow cytometry to analyze the proliferation and apoptosis of ADSCs treated with lidocaine or adrenaline. Results The viable ADSCs yield from liposuction was significantly lower than that from lipectomy, while the apoptosis of cells from liposuction was significantly higher than from lipectomy. The paracrine secretion of the two sources of ADSCs was highest when treated with 10−7 mol/L insulin and 10 ng/mL TGF-α, but there were no significant differences in VEGF, IL-6, IL-8 or HGF levels. The ADSCs from lipectomy migrated faster than those from liposuction, and SOD in the lipectomy group was higher than in the liposuction group, whereas MDA of the lipectomy group was lower than that of the liposuction group. The proliferation ADSCs treated with lidocaine or adrenaline was greatly decreased, while apoptosis was significantly increased, and cytotoxicity of lidocaine or adrenaline to ADSCs was dose-dependent. Conclusions Compared with ADSCs from liposuction, the ADSCs from lipectomy have better biological characteristics. Lidocaine and adrenaline decreased the viability of ADSCs, and their cytotoxicity to ADSCs was dose-dependent. PMID:27610618

  5. Effects of Cx43 gene modification on the proliferation and migration of the human lung squamous carcinoma cell line NCI-H226.

    PubMed

    Zang, J-P; Wei, R

    2015-10-27

    In this study, the human lung squamous carcinoma cell line NCI-H226 was transfected with the recombinant plasmid pBudCE4.1_Cx43 to explore the role of the Cx43 gene in cell growth, cell cycle, and tumor migration. pBudCE4.1-Cx43 was transfected into human lung squamous carcinoma NCI-H226 cells using Lipofectamine TM2000. The mRNA and protein expressions of Cx43 in the transfected cells were detected by reverse transcriptase polymerase chain reaction and western blot analysis. The cell-cell communication was detected using the scratch dye tracer method and the cell cycle was detected by flow cytometry. The CCK-8 proliferation, scratch healing, and cell invasion assays were performed to evaluate the effect of the Cx43 gene transfection on the proliferation, migration, and invasive abilities of NCI-H226 cells. Cx43 mRNA and protein expressions and the fluorescence intensity in the scratch healing test were significantly higher in the experimental group than those in the control and blank groups (P < 0.05 and < 0.01, respectively). The CCK-8 proliferation assay and the scratch healing experiment revealed significantly inhibited NCI-H226 cell proliferation (especially 72 h after incubation) and cell migration, respectively, in the experimental group, compared to the control and blank groups (P < 0.001 and <0.05, respectively). The transwell chamber test showed a statistically significant decrease in the invasive ability of NCI-H226 cells in the experimental group (P < 0.05). Therefore, Cx43 gene transfection could inhibit the migration of human lung squamous carcinoma cell line NCI-H226, thereby inhibiting tumor cell proliferation.

  6. Effects of silencing the ATP-binding cassette protein E1 gene by electroporation on the proliferation and migration of EC109 human esophageal cancer cells.

    PubMed

    Li, Xiao-Rui; Yang, Liu-Zhong; Huo, Xiao-Qing; Wang, Ying; Yang, Qing-Hui; Zhang, Qing-Qin

    2015-07-01

    In the present study, the gene expression of ATP-binding cassette protein E1 (ABCE1) in the EC109 human esophageal cancer cell line was silenced using electroporation to examine the effect if the ABCE1 gene on the growth migration and cell cycle of cancer cells. The small interference (si)RNA sequence of ABCE1 was designed and synthesized to transfect the EC109 cells by electroporation. The mRNA and protein expression levels of ABCE1 were then detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of the cell cycle and apoptosis was performed using flow cytometry. The effect of silencing the ABCE1 gene on the proliferation, migration and invasive ability of the EC109 human esophageal cancer cells were assessed using a Cell counting kit-8 (CCK-8) and with proliferation, wound-healing and cell invasion assays. The mRNA and protein expression levels of ABCE1 were significantly lower in the experimental group compared with the control group (P<0.05). The apoptotic rate of the experimental group was markedly higher than the control group and blank group (P<0.01). The CCK-8 proliferation assay revealed that, compared with the control and blank groups, the proliferation of the EC109 cells in the experimental group was significantly inhibited (P<0.05). The wound healing assay revealed that the migration capacity of the cells in the experimental group was significantly decreased (P<0.05). The Transwell chamber assay demonstrated that the invasive ability of the EC109 cells in the experimental group was significantly decreased (P<0.01). These results revealed that ABCE1 is closely associated with cell proliferation, invasion and migration in esophageal cancer and silencing the ABCE1 gene by electroporation can significantly reduce the proliferation, invasion and migration capacity of EC109 cells in vitro.

  7. [Ginsenoside Rh₂ induces apoptosis and autophagy of K562 cells by activating p38].

    PubMed

    Liu, Xiao-Xia; Xia, Jing; Tang, Jia-Feng; Zhou, Ming-Hua; Chen, Di-Long; Liu, Ze-Hong

    2017-01-01

    To study the effect of ginseng saponin Rh₂ in inducing apoptosis of human leukemia K562 cells, and explore its mechanism from the aspect of autophagy pathway. CCK-8 assay was used to examine the growth inhibition of human leukemia cell lines K562 treated with ginsenoside Rh₂; flow cytometry (FCM) was used to detect cell apoptosis; Hoechst staining was used to observe the changes of cell morphological apoptosis; Acridine and MDC staining were used to detect the effects of the Rh₂ on autophagy; Western blot and RT-PCR were used to detect the expression levels of the proteins closely associated with autophagy and apoptosis. In order to study the effect of autophagy in proliferation and apoptosis, we used the autophagy inhibitor (3-MA).CCK-8 indicated that Rh₂ at low concentration could effectively inhibit the proliferation of leukemia cellsin dose- and time-dependent manners in K562 cells; FCM indicated that Rh₂ induced apoptosis; Hoechest staining showed that K562 cells had typical apoptotic morphological changes by treated Rh₂; Acridine and MDC staining showed that Rh₂ enhanced the green fluorescence and a large number of acidic autophagy vesicles were present; Western blot and RT-PCR results showed that Rh₂ increased the expression levels of Beclin-1, LC3A, LC3B, activated Caspase-3 and p-p38 in K562 cells; application of autophagy inhibitors(3-MA) could weaken the inhibition effect of Rh₂ on proliferation and induction effect on apoptosis in K562 cells. Ginsenoside Rh₂ inhibited the proliferation and induced apoptosis probably through activating p-p38, and inducing cell autophagy signaling pathway in K562 cells. Copyright© by the Chinese Pharmaceutical Association.

  8. Adenosine triphosphate-induced rabbit corneal endothelial cell proliferation in vitro via the P2Y2-PI3K/Akt signaling axis.

    PubMed

    Chen, Junzhao; Shao, Chunyi; Lu, Wenjuan; Yan, Chenxi; Yao, Qinke; Zhu, Mengyu; Chen, Ping; Gu, Ping; Fu, Yao; Fan, Xianqun

    2014-01-01

    To investigate the effect of the ATP-P2Y2-PI3K/Akt signaling axis on promoting rabbit corneal endothelial cell (RCEC) proliferation in vitro. Five concentrations of adenosine triphosphate (ATP; 1, 10, 25, 50 and 100 μM) were added to RCECs, and the cell proliferation was detected using Cell Counting Kit-8 (CCK8) and Ki67 immunohistochemical staining. Other P2Y2 receptor agonists and antagonists were added to the cells, and the proliferation effect was evaluated using CCK8 to determine the involvement of the P2Y2 receptor. Changes in the expression of phosphorylated Akt in RCECs treated with different concentrations of extracellular ATP and the duration of extracellular ATP on Akt phosphorylation were investigated using Western blotting. The pharmacological profiles with or without the PI3K/Akt pathway inhibitors were also determined using Western blotting. We found that 10 μM ATP strongly promoted RCEC proliferation in vitro. Additionally, 25 μM ATP had a proliferation effect, whereas other concentrations (1, 50 and 100 μM) had no effect compared with the control group. Selective P2Y2 receptor agonists (UTP, ATPγS and Ap4A) showed the same promotion effect, while P2Y2 antagonists and PI3K/Akt inhibitors inhibited the effect of ATP. Moreover, phosphorylated Akt could be induced by the addition of extracellular ATP at all five concentrations and lasted for 1 h. This phosphorylation was prevented by PI3K/Akt inhibitors and a P2Y2 antagonist. These findings showed that 10 μM ATP markedly promoted RCEC proliferation via the P2Y2-PI3K/Akt signaling axis. © 2014 S. Karger AG, Basel.

  9. Activation of ALDH2 with Low Concentration of Ethanol Attenuates Myocardial Ischemia/Reperfusion Injury in Diabetes Rat Model

    PubMed Central

    Kang, Pin-Fang; Wu, Wen-Juan; Tang, Yang; Xuan, Ling; Guan, Su-Dong; Tang, Bi; Zhang, Heng

    2016-01-01

    The aim of this paper is to observe the change of mitochondrial aldehyde dehydrogenase 2 (ALDH2) when diabetes mellitus (DM) rat heart was subjected to ischemia/reperfusion (I/R) intervention and analyze its underlying mechanisms. DM rat hearts were subjected to 30 min regional ischemia and 120 min reperfusion in vitro and pretreated with ALDH2 activator ethanol (EtOH); cardiomyocyte in high glucose (HG) condition was pretreated with ALDH2 activator Alda-1. In control I/R group, myocardial tissue structure collapse appeared. Compared with control I/R group, left ventricular parameters, SOD activity, the level of Bcl-2/Bax mRNA, ALDH2 mRNA, and protein expressions were decreased and LDH and MDA contents were increased, meanwhile the aggravation of myocardial structure injury in DM I/R group. When DM I/R rats were pretreated with EtOH, left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 expression were increased; LDH, MDA, and myocardial structure injury were attenuated. Compared with DM + EtOH I/R group, cyanamide (ALDH2 nonspecific blocker), atractyloside (mitoPTP opener), and wortmannin (PI3K inhibitor) groups all decreased left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 and increased LDH, MDA, and myocardial injury. When cardiomyocyte was under HG condition, CCK-8 activity and ALDH2 protein expression were decreased. Alda-1 increased CCK-8 and ALDH2. Our findings suggested enhanced ALDH2 expression in diabetic I/R rats played the cardioprotective role, maybe through activating PI3K and inhibiting mitoPTP opening. PMID:27829984

  10. Uvangoletin induces mitochondria-mediated apoptosis in HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances.

    PubMed

    Zheng, Zhuanzhen; Qiao, Zhenhua; Gong, Rong; Wang, Yalin; Zhang, Yiqun; Ma, Yanping; Zhang, Li; Lu, Yujin; Jiang, Bo; Li, Guoxia; Dong, Chunxia; Chen, Wenliang

    2016-02-01

    This study investigated the cytotoxic effect of uvangoletin on HL-60 cells, and the effects of uvangoletin on myelosuppression, leucopenia, gastrointestinal tract disturbances and the possible cytotoxic mechanisms by using CCK-8, flow cytometry, western blot, xenograft, cyclophosphamide-induced leucopenia, copper sulfate-induced emesis and ethanol-induced gastric mucosal lesions assays. The results of CCK-8, flow cytometry and western blot assays indicated that uvangoletin showed the cytotoxic effect on HL-60 cells and induced the apoptosis of HL-60 cells by downregulating the expression levels of anti-apoptotic proteins (Survivin, Bcl-xl and Bcl-2), upregulating the expression levels of pro-apoptotic proteins (Smac, Bax, Bad, c-caspase-3 and c-caspase-9), and promoting the release of cytochrome c from mitochondria to cytoplasm. Further, the results of xenograft assay suggested that uvangoletin inhibited the HL-60-induced tumor growth without adverse effect on body weight of nude mice in vivo by regulating the expression levels of above apoptotic proteins. The results indicated that the reductions of WBCs count and thighbone marrow granulocytes percentage in cyclophosphamide-induced leucopenia assay, the incubation period and number of emesis in copper sulfate-induced emesis assay and the gastric mucosal lesions in ethanol-induced gastric mucosal lesions assay were not exacerbated or reversed by uvangoletin. In conclusion, the research preliminarily indicated that uvangoletin induced apoptosis of HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances, and the pro-apoptotic mechanisms may be related to mitochondria-mediated apoptotic pathway.

  11. [Mechanism of HL-60 cells apoptosis induced by proteasome inhibitor MG132].

    PubMed

    Zhou, Yong-Ming; Yu, Mei-Xia; Qiu, Yu-Zhen; Xing, Xiao-Lei; Yao, Chun-Hong; Bai, Ru-Jun

    2013-08-01

    The purpose of this study was to elucidate the apoptosis, apoptotic pathway of HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Apoptosis of HL-60 cells was detected by flow cytometry, the expression of P21, P27 and P53 proteins in HL-60 cells treated with MG132 was assayed by Western blot. The HL-60 cells were treated with 1 µmol/L MG132 for 48 h, and irradiated by 75 Gy of (60)Co γ-ray, but their antigenicity was preserved. The effect of irradiated HL-60 cells treated with MG132 on proliferation of peripheral blood mononuclear cells (PBMNC) was measured by CCK-8 method. The results showed that the apoptotic rate of MG132-treated HL-60 cells increased in dose-and time-dependent manner. No significant changes in MG132-induced apoptosis were observed after inhibiting caspase-8 and caspase-9 pathway. The expression of P21 and P27 protein increased after treatment of HL-60 cells with MG132. CCK-8 test showed that HL-60 cells induced with low-dose of MG132 displayed the enhancing effect on proliferation of PBMNC. It is concluded that high dose of MG132 can induce the apoptosis of HL-60 cells, and has direct killing effect on HL-60 cells, but this inducing apoptotic effect on HL-60 cells can not be realized through caspase-8 and caspase-9 pathway. The P21 and P27 protein may be involved in MG132 induced HL-60 cell apoptosis. Low dose of MG132 promotes the proliferation of PBMNC in healthy individuals and enhance the immunity of organism.

  12. Sustained-release genistein from nanostructured lipid carrier suppresses human lens epithelial cell growth

    PubMed Central

    Liu, Jin-Lu; Zhang, Wen-Ji; Li, Xue-Dong; Yang, Na; Pan, Wei-San; Kong, Jun; Zhang, Jin-Song

    2016-01-01

    AIM To design and investigate the efficacy of a modified nanostructured lipid carrier loaded with genistein (Gen-NLC) to inhibit human lens epithelial cells (HLECs) proliferation. METHODS Gen-NLC was made by melt emulsification method. The morphology, particle size (PS), zeta potentials (ZP), encapsulation efficiency (EE) and in vitro release were characterized. The inhibition effect of nanostructured lipid carrier (NLC), genistein (Gen) and Gen-NLC on HLECs proliferation was evaluated by cell counting kit-8 (CCK-8) assay, gene and protein expression of the proliferation marker Ki67 were evaluated with real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence analyses. RESULTS The mean PS of Gen-NLC was 80.12±1.55 nm with a mean polydispersity index of 0.11±0.02. The mean ZP was -7.14±0.38 mV and the EE of Gen in the nanoparticles was 92.3%±0.73%. Transmission electron microscopy showed that Gen-NLC displayed spherical-shaped particles covered by an outer-layer structure. In vitro release experiments demonstrated a prolonged drug release for 72h. The CCK-8 assay results showed the NLC had no inhibitory effect on HLECs and Gen-NLC displayed a much more prominent inhibitory effect on cellular growth compared to Gen of the same concentration. The mRNA and protein expression of Ki67 in LECs decreased significantly in Gen-NLC group. CONCLUSION Sustained drug release by Gen-NLCs may impede HLEC growth. PMID:27275415

  13. Regulation of muscarinic acetylcholine receptors in cultured guinea pig pancreatic acini

    SciTech Connect

    Hootman, S.R.; Brown, M.E.; Williams, J.A.; Logsdon, C.D.

    1986-07-01

    Regulation of muscarinic receptors in cultured guinea pig pancreatic acini was investigated by assessing the effects of cholinergic agonists on binding of (N-methyl-TH)scopolamine ((TH)NMS) and on amylase release. Freshly dispersed acini bound (TH)NMS with a K/sub d/ of 74 pM and a maximal binding level (B/sub max/) of 908 fmol/mg DNA. Carbachol (CCh) stimulated amylase secretion and inhibited (TH)NMS binding. Incubation of acini for 30 min with 0.1 mM CCh decreased the subsequent efficacy of CCh in stimulating amylase release by threefold but had no effect on its potency. In contrast, amylase release in response to cholecystokinin octapeptide (CCK-8) was not altered by CCh preincubation. (TH)NMS binding to acini was decreased only 15-20% after 30-min incubation with CCh. However, culture of acini with 0.1 mM CCh decreased (TH)NMS binding by 50% at 3-4 h and by 85-90% at 24 h. This decrease was attributable primarily to a reduction in B/sub max/ (TH)NMS binding also was decreased to a similar extent by the cholinergic agonists bethanechol and methacholine but not by other secretagogues. The decrease in antagonist binding induced by CCh was dose dependent, with the IC50, 5.8 M, approximating the EC50 for amylase release, 4.3 M. Cultured of acini for 24 h with CCh abolished subsequent amylase release in response to CCh but not to CCK-8. The results indicate that muscarinic receptor turnover in the pancreatic acinus is regulated by receptor activation and that both a decease in receptor numbers and sensitivity to agonists follows prolonged cholinergic agonist exposure.

  14. High fat diet and body weight have different effects on cannabinoid CB(1) receptor expression in rat nodose ganglia.

    PubMed

    Cluny, N L; Baraboi, E D; Mackie, K; Burdyga, G; Richard, D; Dockray, G J; Sharkey, K A

    2013-12-01

    Energy balance is regulated, in part, by the orexigenic signaling pathways of the vagus nerve. Fasting-induced modifications in the expression of orexigenic signaling systems have been observed in vagal afferents of lean animals. Altered basal cannabinoid (CB1) receptor expression in the nodose ganglia in obesity has been reported. Whether altered body weight or a high fat diet modifies independent or additive changes in CB1 expression is unknown. We investigated the expression of CB1 and orexin 1 receptor (OX-1R) in the nodose ganglia of rats fed ad libitum or food deprived (24h), maintained on low or high fat diets (HFD), with differing body weights. Male Wistar rats were fed chow or HFD (diet-induced obese: DIO or diet-resistant: DR) or were body weight matched to the DR group but fed chow (wmDR). CB1 and OX-1R immunoreactivity were investigated and CB1 mRNA density was determined using in situ hybridization. CB1 immunoreactivity was measured in fasted rats after sulfated cholecystokinin octapeptide (CCK8s) administration. In chow rats, fasting did not modify the level of CB1 mRNA. More CB1 immunoreactive cells were measured in fed DIO, DR and wmDR rats than chow rats; levels increased after fasting in chow and wmDR rats but not in DIO or DR rats. In HFD fasted rats CCK8s did not reduce CB1 immunoreactivity. OX-1R immunoreactivity was modified by fasting only in DR rats. These data suggest that body weight contributes to the proportion of neurons expressing CB1 immunoreactivity in the nodose ganglion, while HFD blunts fasting-induced increases, and CCK-induced suppression of, CB1-immunoreactivity. © 2013.

  15. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    PubMed

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  16. Distribution of cholecystokinin receptor binding sites in the human brain: an autoradiographic study

    SciTech Connect

    Dietl, M.M.; Probst, A.; Palacios, J.M.

    1987-01-01

    Cholecystokinin (CCK) binding sites were localized by in vitro autoradiography in human postmortem brain materials from 12 patients without reported neurological diseases using (125I)Bolton-Hunter CCK octapeptide (BHCCK-8) as a ligand. The pharmacological characteristics of BHCCK-8 binding to mounted tissue sections were comparable to those previously reported in the rat. CCK-8 being the most potent displacer, followed by caerulein, CCK-4, and gastrin I. The distribution of BHCCK-8 binding sites was heterogeneous. These sites were highly concentrated in a limited number of gray matter areas and nuclei. The highest binding densities were seen in the glomerular and external plexiform layers of the olfactory bulb. BHCCK-8 binding sites were also enriched in the neocortex, where they presented a laminar distribution with low levels in lamina I, moderate concentration in laminae II to IV, high density in lamina V, and low levels in lamina VI. A different laminar distribution was seen in the visual cortex, where a low receptor density was observed in lamina IV but higher density in laminae II and VI. In the basal ganglia the nucleus accumbens, caudatus, and the putamen presented moderate to high densities of binding sites, while the globus pallidus lacked sites of BHCCK-8 binding. In the limbic system the only area presenting moderate to high density was the amygdaloid complex, particularly in the granular nucleus, while most of the thalamic nuclei were extremely poor or lacked BHCCK-8 binding. The hippocampal formation showed low (CA1-3) to moderate (subiculum) densities. Midbrain areas generally disclosed very low levels of BHCCK-8 binding sites. The pontine gray and the nucleus reticularis tegmenti pontis showed a relatively high density of CCK-8 receptor specific binding.

  17. Activation of ALDH2 with Low Concentration of Ethanol Attenuates Myocardial Ischemia/Reperfusion Injury in Diabetes Rat Model.

    PubMed

    Kang, Pin-Fang; Wu, Wen-Juan; Tang, Yang; Xuan, Ling; Guan, Su-Dong; Tang, Bi; Zhang, Heng; Gao, Qin; Wang, Hong-Ju

    2016-01-01

    The aim of this paper is to observe the change of mitochondrial aldehyde dehydrogenase 2 (ALDH2) when diabetes mellitus (DM) rat heart was subjected to ischemia/reperfusion (I/R) intervention and analyze its underlying mechanisms. DM rat hearts were subjected to 30 min regional ischemia and 120 min reperfusion in vitro and pretreated with ALDH2 activator ethanol (EtOH); cardiomyocyte in high glucose (HG) condition was pretreated with ALDH2 activator Alda-1. In control I/R group, myocardial tissue structure collapse appeared. Compared with control I/R group, left ventricular parameters, SOD activity, the level of Bcl-2/Bax mRNA, ALDH2 mRNA, and protein expressions were decreased and LDH and MDA contents were increased, meanwhile the aggravation of myocardial structure injury in DM I/R group. When DM I/R rats were pretreated with EtOH, left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 expression were increased; LDH, MDA, and myocardial structure injury were attenuated. Compared with DM + EtOH I/R group, cyanamide (ALDH2 nonspecific blocker), atractyloside (mitoPTP opener), and wortmannin (PI3K inhibitor) groups all decreased left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 and increased LDH, MDA, and myocardial injury. When cardiomyocyte was under HG condition, CCK-8 activity and ALDH2 protein expression were decreased. Alda-1 increased CCK-8 and ALDH2. Our findings suggested enhanced ALDH2 expression in diabetic I/R rats played the cardioprotective role, maybe through activating PI3K and inhibiting mitoPTP opening.

  18. Involvement of autophagy in tantalum nanoparticle-induced osteoblast proliferation.

    PubMed

    Kang, Chengrong; Wei, Limin; Song, Bin; Chen, Liangjiao; Liu, Jia; Deng, Bin; Pan, Xuan; Shao, Longquan

    2017-01-01

    Porous tantalum (Ta) implants are highly corrosion resistant and biocompatible, and they possess significantly better initial stability than that of conventional titanium (Ti) implants. During loading wear, Ta nanoparticles (Ta-NPs) that were deposited on the surface of a porous Ta implant are inevitably released and come into direct contact with peri-implant osteoblasts. The wear debris may influence cell behavior and implant stabilization. However, the interaction of Ta-NPs with osteoblasts has not been clearly investigated. This study aimed to investigate the effect of Ta-NPs on cell proliferation and their underlying mechanism. The Cell Counting Kit-8 (CCK-8) assay was used to measure the cell viability of MC3T3-E1 mouse osteoblasts and showed that Ta-NP treatment could increase cell viability. Then, confocal microscopy, Western blotting, and transmission electron microscopy were used to confirm the autophagy induced by Ta-NPs, and evidence of autophagy induction was observed as positive LC3 puncta, high-LC3-II expression, and autophagic vesicle ultrastructures. The CCK-8 assay revealed that the cell viability was further increased and decreased by the application of an autophagy inducer and inhibitor, respectively. In addition, pre-treatment with autophagy inhibitor 3-methyladenine (3-MA) inhibited the Ta-NP-induced autophagy. These results indicate that the Ta-NPs can promote cell proliferation, that an autophagy inducer can further strengthen this effect and that an autophagy inhibitor can weaken this effect. In conclusion, autophagy was involved in Ta-NP-induced cell proliferation and had a promoting effect.

  19. Smilax china L. rhizome extract inhibits nuclear factor-κB and induces apoptosis in ovarian cancer cells.

    PubMed

    Hu, Li-ling; Chen, Dong-sheng; Wang, Yan-yan; Qin, You; Huang, Pu; Yu, Li-xiu; Liao, Jing; Hua, Xiao-li

    2015-12-01

    To study the antitumor effects and associated mechanisms of extract of the Smilax china L. rhizome (SCR) on ovarian cancer cells. Ovarian cancer cells A2780 were treated with different concentrations of SCR extract (SCRE), and compared with controls. Effects on cell growth were evaluated by cell counting kit-8 (CCK-8) assay; proliferation effects by EdU incorporation assay; cell cycle by propidium iodide staining; apoptosis by annexin V-fluorescein isothiocyanate/propidium iodide; cellular distribution of nuclear factor-κB (NF-κB) by immunofluorescence; protein levels of NF-κB, caspase-3, poly-adenosine diphosphate (ADP)-ribose polymerase (PARP), Bcl-2-associated X protein (Bax), cellular inhibitor of apoptosis (cIAP)-1, anti-X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-extra large (Bcl-XL), B-cell lymphoma-2 (Bcl-2) and AKT by Western blotting; and effects of SCRE combined with cisplatin or adriamycin on A2780 cells by CCK-8 assay. SCRE suppressed A2780 cell proliferation in a dose-dependent manner (P<0.05,P<0.01), arrested cells in G2/M phase and induced apoptosis by activating caspase-3, PARP and Bax. SCRE treatment also correlated with inhibition of NF-κB and downregulation of Bcl-2, Bcl-XL, cIAP-1, XIAP and AKT. SCRE can promote chemosensitivity to cisplatin and adriamycin in A2780 cells (P<0.01). SCR effectively inhibits NF-κB, induces apoptosis and reduces chemoresistance to cisplatin and adriamycin in ovarian cancer cells, which might be its molecular basis for treating ovarian cancer.

  20. Effects of tacrolimus on morphology, proliferation and differentiation of mesenchymal stem cells derived from gingiva tissue

    PubMed Central

    HA, DONG-HO; YONG, CHUL SOON; KIM, JONG OH; JEONG, JEE-HEON; PARK, JUN-BEOM

    2016-01-01

    Tacrolimus is a 23-membered macrolide lactone with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following kidney, heart and liver transplantation. Tacrolimus also exerts a variety of actions on bone metabolism. The aim of the present study was to evaluate the effects of different concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva. Gingival-derived stem cells were grown in the presence of tacrolimus at final concentrations ranging from 0.001 to 100 µg/ml. The morphology of the cells was viewed under an inverted microscope and the cell viability was analyzed using Cell Counting kit-8 (CCK-8) on days 1, 3, 5 and 7. Alizarin Red S staining was used to assess mineralization of treated cells. The control group showed spindle-shaped, fibroblast-like morphology and the shapes of the cells in 0.001, 0.01, 0.1, 1 and 10 µg/ml tacrolimus were similar to those of the control group. All groups except the 100 µg/ml group showed increased cell proliferation over time. Cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, 1 and 10 µg/ml were not identified to be significantly different compared with the control at days 1, 3 and 5 using the CCK-8 assays. Increased mineralized deposits were noted with increased incubation time. Treatment with tacrolimus from 0.001 to 1 µg/ml led to an increase in mineralization compared with the control group. Within the limits of this study, tacrolimus at the tested concentrations (ranging from 0.001 to 10 µg/ml) did not result in differences in the viability of stem cells derived from gingiva; however it did enhance osteogenic differentiation of the stem cells. PMID:27177273

  1. Influence of Cu content on the cell biocompatibility of Ti-Cu sintered alloys.

    PubMed

    Zhang, Erlin; Zheng, Lanlan; Liu, Jie; Bai, Bing; Liu, Cong

    2015-01-01

    The cell toxicity and the cell function of Ti-Cu sintered alloys with different Cu contents (2, 5, 10 and 25 wt.%, respectively) have been investigated in comparison with commercial pure titanium in order to assess the influence of Cu content on the cell biocompatibility of the Ti-Cu alloys. The cytotoxicity was studied by examining the MG63 cell response by CCK8 assessment. The cell morphology was evaluated by acridine orange/ethidium bromide (AO/EB) fluorescence and observed under scanning electronic microscopy (SEM). The cell function was monitored by measuring the AKP activity. It has been shown by the AO/EB morphology results that the cell death on both cp-Ti sample and Ti-Cu samples is due to apoptosis rather than necrosis. Although more apoptotic cells were found on the Ti-2Cu and Ti-5Cu samples, no evidence of Cu content dependent manner of apoptosis has been found. SEM observation indicated very good cell adhesion and spread on the cp-Ti sample and the Ti-Cu samples with different Cu contents. CCK8 results displayed that increase in the Cu content in Ti-Cu alloys does not bring about any difference in the cell viability. In addition, AKP test results indicated that no difference in the differentiation of MG63 was found between the cp-Ti and the Ti-Cu samples and among the Ti-Cu samples. All results indicated that Ti-Cu alloys exhibit very good cell biocompatibility and the Cu content up to 25 wt.% in the Ti-Cu alloys has no influence on the cell proliferation and differentiation.

  2. Can graphene oxide cause damage to eyesight?

    PubMed

    Yan, Lu; Wang, Yaping; Xu, Xu; Zeng, Chao; Hou, Jiangping; Lin, Mimi; Xu, Jingzhou; Sun, Fei; Huang, Xiaojie; Dai, Liming; Lu, Fan; Liu, Yong

    2012-06-18

    As graphene becomes one of the most exciting candidates for multifunctional biomedical applications, contact between eyes and graphene-based materials is inevitable. On the other hand, eyes, as a special organ in the human body, have unique advantages to be used for testing new biomedical research and development, such as drug delivery. Intraocular biocompatible studies on graphene-related materials are thus essential. Here, we report our recent studies on intraocular biocompatibility and cytotoxicity of graphene oxide (GO) both in vitro and in vivo. The successful preparation of GO nanosheets was confirmed using atomic force microscopy, contact angle analyzer, Fourier transform infrared spectroscopy, and Raman spectroscopy. The influence of GO on human retinal pigment epithelium (RPE) cells in terms of the cell morphology, viability, membrane integrity, and apoptosis was investigated using various techniques, including optical micrography, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, and apoptosis assay. The addition of GO had little influence on cell morphology, but the change was visible after long-time culturing. RPE cells showed higher than 60% cell viability by CCK-8 assay in GO solutions and less than 8% LDH release, although a small amount of apoptosis (1.5%) was observed. In vitro results suggested good biocompatibility of GO to RPE cells with slight adverse influence, on the cell viability and morphology in long-time periods, along with aggregation of GO. Thus, some further studies are needed to clarify the cytotoxicity mechanism of GO. GO intravitreally injected eyes showed few changes in eyeball appearance, intraocular pressure (IOP), eyesight, and histological photos. Our results suggested that GO did not cause any significant toxicity to the cell growth and proliferation. Intravitreal injection of GO into rabbits' eyes did not lead to much change in the eyeball appearance, IOP, electroretinogram, and histological examination.

  3. Metformin Inhibits TGF-β1-Induced Epithelial-to-Mesenchymal Transition via PKM2 Relative-mTOR/p70s6k Signaling Pathway in Cervical Carcinoma Cells

    PubMed Central

    Cheng, Keyan; Hao, Min

    2016-01-01

    Background: Epithelial-to-mesenchymal transition (EMT) plays a prominent role in tumorigenesis. Metformin exerts antitumorigenic effects in various cancers. This study investigated the mechanisms of metformin in TGF-β1-induced Epithelial-to-mesenchymal transition (EMT) in cervical carcinoma cells. Methods: cells were cultured with 10 ng/mL TGF-β1 to induce EMT and treated with or without metformin. Cell viability was evaluated by CCK-8 (Cell Counting Kit 8, CCK-8) assay; apoptosis were analyzed by flow cytometry; cell migration was evaluated by wound-healing assay. Western blotting was performed to detect E-cadherin, vimentin, signal transducer and activator of transcription 3 (STAT3), snail family transcriptional repressor 2 (SNAIL2), phosphorylation of p70s6k (p-p70s6k) and -Pyruvate kinase M2 (PKM2) Results: TGF-β1 promoted proliferation and migration, and it attenuated apoptosis compared with cells treated with metformin with or without TGF-β1 in cervical carcinoma cells. Moreover, metformin partially abolished TGF-β1-induced EMT cell proliferation and reversed TGF-β1-induced EMT. In addition, the anti-EMT effects of metformin could be partially in accord with rapamycin, a specific mTOR inhibitor. Metformin decreased the p-p70s6k expression and the blockade of mTOR/p70s6k signaling decreased PKM2 expression. Conclusion: Metformin abolishes TGF-β1-induced EMT in cervical carcinoma cells by inhibiting mTOR/p70s6k signaling to down-regulate PKM2 expression. Our study provides a novel mechanistic insight into the anti-tumor effects of metformin. PMID:27916907

  4. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

    PubMed

    Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

    2015-11-01

    Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

  5. Three-dimensional Printed Scaffolds with Gelatin and Platelets Enhance In vitro Preosteoblast Growth Behavior and the Sustained-release Effect of Growth Factors

    PubMed Central

    Zhu, Wei; Xu, Chi; Ma, Bu-Peng; Zheng, Zhi-Bo; Li, Yu-Long; Ma, Qi; Wu, Guo-Liang; Weng, Xi-Sheng

    2016-01-01

    Background: Three-dimensional (3D) printing technology holds great promise for treating diseases or injuries that affect human bones with enhanced performance over traditional techniques. Different patterns of design can lead to various mechanical properties and biocompatibility to various degrees. However, there is still a long way to go before we can fully take advantage of 3D printing technologies. Methods: This study tailored 3D printed scaffolds with gelatin and platelets to maximize bone regeneration. The scaffolds were designed with special internal porous structures that can allow bone tissue and large molecules to infiltrate better into the scaffolds. They were then treated with gelatin and platelets via thermo-crosslinking and freeze-drying, respectively. Vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1 were measured at different time points after the scaffolds had been made. Cell proliferation and cytotoxicity were determined via cell counting kit-8 (CCK-8) assay. Results: There was a massive boost in the level of VEGF and TGF-β1 released by the scaffolds with gelatin and platelets compared to that of scaffolds with only gelatin. After 21 days of culture, the CCK-8 cell counts of the control group and treated group were significantly higher than that of the blank group (P < 0.05). The cytotoxicity test also indicated the safety of the scaffolds. Conclusions: Our experiments confirmed that the 3D printed scaffolds we had designed could provide a sustained-release effect for growth factors and improve the proliferation of preosteoblasts with little cytotoxicity in vitro. They may hold promise as bone graft substitute materials in the future. PMID:27779164

  6. Characterization of the binding of (3H)-(+/-)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors

    SciTech Connect

    Chang, R.S.; Lotti, V.J.; Chen, T.B.; Kunkel, K.A.

    1986-09-01

    (3H)-(+/-)-L-364,718 a new, potent and selective nonpeptide peripheral cholecystokinin (CCK) antagonist bound saturably and reversibly to rat pancreatic membranes. The radioligand recognized a single class of binding sites with a high affinity (Kd = 0.23 nM). The binding of (/sup 3/H)-(+/-)-L-364,718 was stereospecific in that the more biologically active (-)-enantiomer demonstrated greater potency than the (+)-enantiomer. The rank order of potency of various CCK agonists and antagonists in displacing (/sup 3/H)-(+/-)-L-364,718 correlated with their ability to displace (/sup 125/I)CCK-8 and their known pharmacological activities in peripheral tissues. However, the absolute potencies of agonists were greater in displacing (/sup 125/I)CCK-8 than (/sup 3/H)-(+/-)-L-364,718. As described for other physiologically relevant receptor systems, the potency for displacement of (/sup 3/H)-(+/-)-L-364,718 binding by CCK agonists, but not antagonists, was reduced by guanosine 5'-(beta, gamma-imido)triphosphate and NaCl and enhanced by MgCl/sub 2/. (/sup 3/H)-(+/-)-L-364,718 also demonstrated specific binding to bovine gall bladder tissue but not guinea pig brain or gastric glands, consistent with its selectivity as a peripheral CCK antagonist. (/sup 3/H)-(+/-)-L-364,718 binding to pancreatic membranes was not affected by various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. These data indicate that (/sup 3/H)-(+/-)-L-364,718 represents a new potent nonpeptide antagonist radioligand for the study of peripheral CCK receptors which may allow differentiation of agonist and antagonist interactions.

  7. Thrombocytopenia in dogs induced by granulocyte-macrophage colony-stimulating factor: increased destruction of circulating platelets.

    PubMed

    Nash, R A; Burstein, S A; Storb, R; Yang, W; Abrams, K; Appelbaum, F R; Boone, T; Deeg, H J; Durack, L D; Schuening, F G

    1995-09-01

    Administration of recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose-dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In-labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM-CSF-treated dogs. In the

  8. Preparation of 111In-DTPA morpholino oligomer for low abdominal accumulation

    PubMed Central

    Liu, Guozheng; Dou, Shuping; Rusckowski, Mary; Greiner, Dale; Hnatowich, Donald

    2010-01-01

    An ability to quantitate the beta cell mass by noninvasive nuclear imaging will be very useful in the prevention, diagnosis, and treatment of diabetes. However, to be successful, radioactivity from the pancreas must not be obscured by the background radioactivity in the abdomen. Pretargeting offers the promise of achieving high target organ to normal tissue ratios. In preparation for pancreas imaging studies by pretargeting using morpholino oligomers (MORF/cMORF), it was necessary to develop a simple and efficient method to radiolabel the cMORF effector. Because we have shown that labeling the cMORF with 111In via DTPA reduces excretion into the intestines compared to labeling with 99mTc via MAG3, the conjugation of DTPA to cMORF were investigated for the 111In labeling. The amine-derivatized cMORF was conjugated with DTPA using EDC (1-Ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride) as an alternative to the conventional cyclic anhydride. The conjugation efficiency (represented by the number of DTPA groups attached per cMORF) was investigated by changing the EDC, DTPA and cMORF molar ratios. Different open columns were considered for the purification of DTPA-cMORF. Before conjugation, each cMORF molecule was confirmed to have an amine by trinitrobenzene sulfonic acid (TNBS) assay using the ω-amino butyric acid as positive standrad and the non-amine derivatized cMORF as negative standard. The average number of DTPA groups per cMORF was 0.15–0.20 following the conjugation with DTPA over a cMORF/DTPA molar ratio of 0.5 to 5 and over a cMORF/EDC molar ratio of 20 to 60. The conjugation efficiency was lower than expected probably due to steric hindrance. A 1×50 cm P-4 column using ammonia acetate as eluting buffer provided an adequate separation of DTPA-cMORF from free DTPA. The 111In labeling efficiency by transchelation from acetate exceeded 95%, thus avoiding the need for postlabeling purification. Despite the lower than expected conjugation efficiency

  9. Easy and efficient (111)indium labeling of long-term stored DTPA conjugated protein.

    PubMed

    Nalla, Amarnadh; Buch, Inge; Hesse, Birger

    2011-01-01

    The labelling efficiency of long-term stored DTPA-conjugates has not been reported previously even though DTPA has been in extensive use as metal chelator in the development of radiopharmaceuticals and contrast agents. DTPA is often used as a bifunctional chelating agent conjugated to tumor targeting vehicles such as monoclonal antibodies and receptor directed peptides. The purpose of this study was to monitor the labelling efficiency of a DTPA-conjugate on long-term storage using 111In-chloride at two different temperatures and incubation times for the In-labelling. Cyclic-diethylene-triamine-pentaacetic acid (cDTAP) was conjugated to a polyclonal immunoglobulin-G (IgG) in borate buffer, pH 8.2 at +4?C for 4 hours. Then the DTPA-conjugate was dialyzed against 50 mmol/l sodium citrate buffer saline, pH 6.0 and stored at -80° C in aliquots of 1 mg/0.5 ml. The DTPA-conjugate was labeled with 111In-chloride in citrate buffer, pH 6. The labelling reaction was incubated at room temperature (RT) for 30 min and at +4?C for 90 min. Determination of labelling efficiency was performed using ITLC and an instant chromatography scanner equipped with a NaI crystal. The labelling efficiency of the DTPA-conjugate was monitored every third month for 12 months. The median labelling efficiencies varied between 92 and 96% during the whole period. The two combinations of incubation times and temperatures (30 min at RT and 90 min at +4°C) had no affect on labelling efficiency of the DTPA-conjugate, stored for 12 months. Our study shows that 111In-labelling can easily be performed within 30 min at RT for thermo-stable proteins like polyclonal, DTPA-conjugated IgG stored long-term at -80°C with a high 111In-labelling efficiency.

  10. Preparation of (111)In-DTPA morpholino oligomer for low abdominal accumulation.

    PubMed

    Liu, Guozheng; Dou, Shuping; Rusckowski, Mary; Greiner, Dale; Hnatowich, Donald

    2010-09-01

    An ability to quantitate the beta cell mass by noninvasive nuclear imaging will be very useful in the prevention, diagnosis, and treatment of diabetes. However, to be successful, radioactivity from the pancreas must not be obscured by the background radioactivity in the abdomen. Pretargeting offers the promise of achieving high target organ to normal tissue ratios. In preparation for pancreas imaging studies by pretargeting using morpholino oligomers (MORF/cMORF), it was necessary to develop a simple and efficient method to radiolabel the cMORF effector. Because we have shown that labeling the cMORF with (111)In via DTPA reduces excretion into the intestines compared to labeling with (99m)Tc via MAG(3), the conjugation of DTPA to cMORF was investigated for (111)In labeling. The amine-derivatized cMORF was conjugated with DTPA using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as an alternative to the conventional cyclic anhydride. The conjugation efficiency (represented by the number of DTPA groups attached per cMORF) was investigated by changing the EDC, DTPA, and cMORF molar ratios. Different open columns were considered for the purification of DTPA-cMORF. Before conjugation, each cMORF molecule was confirmed to have an amine by trinitrobenzene sulfonic acid (TNBS) assay using the omega-amino butyric acid as positive standard and the non-amine derivatized cMORF as negative standard. The average number of DTPA groups per cMORF was 0.15-0.20 following the conjugation over a cMORF/DTPA molar ratio of 0.5-5 and over a cMORF/EDC molar ratio of 20-60. The conjugation efficiency was lower than expected probably due to steric hindrance. A 1 x 50cm P-4 column using ammonium acetate as eluting buffer provided an adequate separation of DTPA-cMORF from free DTPA. The (111)In labeling efficiency by transchelation from acetate exceeded 95%, thus avoiding the need for postlabeling purification. Despite the lower than expected conjugation efficiency in which

  11. Biodistribution of indium-111-labeled OC 125 monoclonal antibody after intraperitoneal injection in nude mice intraperitoneally grafted with ovarian carcinoma

    SciTech Connect

    Thedrez, P.; Saccavini, J.C.; Nolibe, D.; Simoen, J.P.; Guerreau, D.; Gestin, J.F.; Kremer, M.; Chatal, J.F. )

    1989-06-01

    The purpose of this work was to study the biodistribution of 111In-labeled OC 125 monoclonal antibody (MAb) with known affinity for ovarian carcinomas in a nude mouse model grafted i.p. with a human ovarian cancer (NIH:OVCAR-3). Tumor uptake 24 h after i.p. injection was higher with intact 111In-labeled OC 125 MAb than with 111In-nonspecific immunoglobulin. The kinetics of tumor uptake also differed, showing a plateau followed by a drop at Day 7 with 111In-OC 125 MAb and a decrease beginning at 24 h with 111In-nonspecific immunoglobulin. Tumor-to-normal tissue ratios ranged between 29.91 +/- 11.85 and 0.68 +/- 0.15 with 111In-OC 125 MAb and between 4.50 +/- 1.06 and 0.53 +/- 0.04 with 111In-nonspecific immunoglobulin according to the normal tissues and the time points considered. Tumor uptake 2 h after injection was the same for F(ab')2 fragments as for intact MAb, whereas maximum uptake at 24 h was lower and was followed by a decrease at Day 4. Tumor-to-normal tissue ratios were in the same range, except for the tumor to blood ratio which was higher and the tumor to kidney ratio which was lower at 24 and 96 h. Maximum tumor uptake was higher after i.p. than i.v. injection. Instead of attaining the plateau noted after i.p. injection, tumor uptake after i.v. injection remained low at 2 h, reaching its peak only after 96 h. 131I-OC 125 injected i.p., which reached maximum tumor uptake at 2 h, showed tumor-to-tissue ratios ranging between 15.98 +/- 2.63 and 0.96 +/- 0.86, i.e., not very different from those with 111In. After i.p. injection of a radiolabeled colloid solution, maximum tumor uptake was reached at 96 h, but with very high nonspecific uptake in liver and spleen. These results indicate high, selective tumor uptake of 111In-OC 125 after i.p.

  12. Neuroendocrine system of the digestive tract in Rhamdia quelen juvenile: an immunohistochemical study.

    PubMed

    Hernández, D R; Vigliano, F A; Sánchez, S; Bermúdez, R; Domitrovic, H A; Quiroga, M I

    2012-08-01

    In this work, an immunohistochemical study was performed to determine the distribution and relative frequencies of some neuromodulators of the digestive tract of silver catfish (Rhamdia quelen). The digestive tract of silver catfish was divided into six portions; the oesophagus, stomach, intestine (ascendant, descendant and convoluted segments), and rectum. Immunohistochemical method using a pool of specific antisera against-gastrin, -cholecystokinin-8, -leu-enkephalin, -neuropeptide Y, -calcitonin gene-related peptide (CGRP), and -vasoactive intestinal peptide (VIP) was employed. Immunoreactivity to all antisera was identified in neuroendocrine cells (NECs) localized in the gut epithelium, although no reaction was observed in the oesophagus or stomach. The morphology of NECs immunopositive to each antibody was similar. They were slender in shape, with basally located nucleus, and their main axis perpendicular to the basement membrane. The number of NECs immunoreactive to all antisera was higher in the ascendant and descendant intestine, exhibiting a decreasing trend toward distal segments of the gut. In addition, immunoreactivity to CGRP and VIP was observed in the myenteric plexus and nerve fibers distributed in the mucosal, submucosal and muscular layers. The higher number of immunopositive NECs in the ascendant and descendant intestine may indicate the primary role of these segments in the control of food intake by means of orexigenic and anorexigenic peripheral signals.

  13. Peripheral oxytocin activates vagal afferent neurons to suppress feeding in normal and leptin-resistant mice: a route for ameliorating hyperphagia and obesity.

    PubMed

    Iwasaki, Yusaku; Maejima, Yuko; Suyama, Shigetomo; Yoshida, Masashi; Arai, Takeshi; Katsurada, Kenichi; Kumari, Parmila; Nakabayashi, Hajime; Kakei, Masafumi; Yada, Toshihiko

    2015-03-01

    Oxytocin (Oxt), a neuropeptide produced in the hypothalamus, is implicated in regulation of feeding. Recent studies have shown that peripheral administration of Oxt suppresses feeding and, when infused subchronically, ameliorates hyperphagic obesity. However, the route through which peripheral Oxt informs the brain is obscure. This study aimed to explore whether vagal afferents mediate the sensing and anorexigenic effect of peripherally injected Oxt in mice. Intraperitoneal Oxt injection suppressed food intake and increased c-Fos expression in nucleus tractus solitarius to which vagal afferents project. The Oxt-induced feeding suppression and c-Fos expression in nucleus tractus solitarius were blunted in mice whose vagal afferent nerves were blocked by subdiaphragmatic vagotomy or capsaicin treatment. Oxt induced membrane depolarization and increases in cytosolic Ca(2+) concentration ([Ca(2+)]i) in single vagal afferent neurons. The Oxt-induced [Ca(2+)]i increases were markedly suppressed by Oxt receptor antagonist. These Oxt-responsive neurons also responded to cholecystokinin-8 and contained cocaine- and amphetamine-regulated transcript. In obese diabetic db/db mice, leptin failed to increase, but Oxt increased [Ca(2+)]i in vagal afferent neurons, and single or subchronic infusion of Oxt decreased food intake and body weight gain. These results demonstrate that peripheral Oxt injection suppresses food intake by activating vagal afferent neurons and thereby ameliorates obesity in leptin-resistant db/db mice. The peripheral Oxt-regulated vagal afferent neuron provides a novel target for treating hyperphagia and obesity. Copyright © 2015 the American Physiological Society.

  14. OATP1B3 is expressed in pancreatic β-islet cells and enhances the insulinotropic effect of the sulfonylurea derivative glibenclamide.

    PubMed

    Meyer Zu Schwabedissen, Henriette E; Boettcher, Kerstin; Steiner, Tobias; Schwarz, Ute I; Keiser, Markus; Kroemer, Heyo K; Siegmund, Werner

    2014-02-01

    Organic anion transporting polypeptide OATP1B3 is a membrane-bound drug transporter that facilitates cellular entry of a variety of substrates. Most of the previous studies focused on its hepatic expression and function in hepatic drug elimination. In this study, we report expression of OATP1B3 in human pancreatic tissue, with the abundance of the transporter localized in the islets of Langerhans. Transport studies using OATP1B3-overexpressing MDCKII cells revealed significant inhibition of the cellular uptake of the known substrate cholecystokinin-8 in the presence of the insulinotropic antidiabetes compounds tolbutamide, glibenclamide, glimepiride, and nateglinide and identified glibenclamide as a novel substrate of OATP1B3. Sulfonylurea derivatives exert their insulinotropic effect by binding to the SUR1 subunit of the KATP channels inducing insulin secretion in β-cells. Here, we show that transient overexpression of human OATP1B3 in a murine β-cell line (MIN6)-which exhibits glucose and glibenclamide-sensitive insulin secretion-significantly enhances the insulinotropic effect of glibenclamide without affecting glucose-stimulated insulin secretion. Taken together, our data provide evidence that the drug transporter OATP1B3 functions as a determinant of the insulinotropic effect of glibenclamide on the tissue level. Changes in transport activity based on drug-drug interactions or genetic variability may therefore influence glibenclamide efficacy.

  15. The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells

    PubMed Central

    Voronina, Svetlana; Collier, David; Chvanov, Michael; Middlehurst, Ben; Beckett, Alison J.; Prior, Ian A.; Criddle, David N.; Begg, Malcolm; Mikoshiba, Katsuhiko; Sutton, Robert; Tepikin, Alexei V.

    2014-01-01

    The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca2+ concentration ([Ca2+]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as ‘initiating’ organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca2+ influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca2+ influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca2+ entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca2+ pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca2+ elevation and endocytic vacuole formation. PMID:25370603

  16. An immunohistochemical study of the gut neuroendocrine system in juvenile pejerrey Odontesthes bonariensis (Valenciennes).

    PubMed

    Vigliano, F A; Muñoz, L; Hernández, D; Cerutti, P; Bermúdez, R; Quiroga, M I

    2011-03-01

    In this study, several neuropeptides were identified by immunohistochemistry in neuroendocrine cells (NEC) located in the gut epithelium and nerve cell bodies of the enteric nervous system of pejerrey Odontesthes bonariensis, a species that is a promising candidate for intensive aquaculture. The neuropeptides involved in orexigenic or anorexigenic action, i.e. gastrin, cholecystokinin-8, neuropeptide Y and calcitonin gene-related peptide (CGRP), displayed a significantly higher number of immunoreactive NECs in the anterior intestine, suggesting that this region of the gut plays an important role in the peripheral control of food intake. On the other hand, leu-enkephalin and vasoactive intestinal peptide (VIP), both associated with the modulation of the enteric immune system, showed no significant variations in the mean value of immunopositive NECs between the anterior and posterior intestine. This may indicate that their activity is required at a similar level along the entire gut. In addition, CGRP and VIP-immunoreactive neurons and nerve fibres were observed in the myenteric plexus, which might exert synergistic effects with the neuropeptides immunolocalized in NECs. © 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.

  17. Chronic CNS oxytocin signaling preferentially induces fat loss in high-fat diet-fed rats by enhancing satiety responses and increasing lipid utilization.

    PubMed

    Blevins, James E; Thompson, Benjamin W; Anekonda, Vishwanath T; Ho, Jacqueline M; Graham, James L; Roberts, Zachary S; Hwang, Bang H; Ogimoto, Kayoko; Wolden-Hanson, Tami; Nelson, Jarrell; Kaiyala, Karl J; Havel, Peter J; Bales, Karen L; Morton, Gregory J; Schwartz, Michael W; Baskin, Denis G

    2016-04-01

    Based largely on a number of short-term administration studies, growing evidence suggests that central oxytocin is important in the regulation of energy balance. The goal of the current work is to determine whether long-term third ventricular (3V) infusion of oxytocin into the central nervous system (CNS) is effective for obesity prevention and/or treatment in rat models. We found that chronic 3V oxytocin infusion between 21 and 26 days by osmotic minipumps both reduced weight gain associated with the progression of high-fat diet (HFD)-induced obesity and elicited a sustained reduction of fat mass with no decrease of lean mass in rats with established diet-induced obesity. We further demonstrated that these chronic oxytocin effects result from 1) maintenance of energy expenditure at preintervention levels despite ongoing weight loss, 2) a reduction in respiratory quotient, consistent with increased fat oxidation, and 3) an enhanced satiety response to cholecystokinin-8 and associated decrease of meal size. These weight-reducing effects persisted for approximately 10 days after termination of 3V oxytocin administration and occurred independently of whether sucrose was added to the HFD. We conclude that long-term 3V administration of oxytocin to rats can both prevent and treat diet-induced obesity.

  18. Beneficial effect of the bioflavonoid quercetin on cholecystokinin-induced mitochondrial dysfunction in isolated rat pancreatic acinar cells.

    PubMed

    Weber, Heike; Jonas, Ludwig; Wakileh, Michael; Krüger, Burkhard

    2014-03-01

    The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca(2+)-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP.

  19. Chronic CNS oxytocin signaling preferentially induces fat loss in high-fat diet-fed rats by enhancing satiety responses and increasing lipid utilization

    PubMed Central

    Thompson, Benjamin W.; Anekonda, Vishwanath T.; Ho, Jacqueline M.; Graham, James L.; Roberts, Zachary S.; Hwang, Bang H.; Ogimoto, Kayoko; Wolden-Hanson, Tami; Nelson, Jarrell; Kaiyala, Karl J.; Havel, Peter J.; Bales, Karen L.; Morton, Gregory J.; Schwartz, Michael W.; Baskin, Denis G.

    2016-01-01

    Based largely on a number of short-term administration studies, growing evidence suggests that central oxytocin is important in the regulation of energy balance. The goal of the current work is to determine whether long-term third ventricular (3V) infusion of oxytocin into the central nervous system (CNS) is effective for obesity prevention and/or treatment in rat models. We found that chronic 3V oxytocin infusion between 21 and 26 days by osmotic minipumps both reduced weight gain associated with the progression of high-fat diet (HFD)-induced obesity and elicited a sustained reduction of fat mass with no decrease of lean mass in rats with established diet-induced obesity. We further demonstrated that these chronic oxytocin effects result from 1) maintenance of energy expenditure at preintervention levels despite ongoing weight loss, 2) a reduction in respiratory quotient, consistent with increased fat oxidation, and 3) an enhanced satiety response to cholecystokinin-8 and associated decrease of meal size. These weight-reducing effects persisted for approximately 10 days after termination of 3V oxytocin administration and occurred independently of whether sucrose was added to the HFD. We conclude that long-term 3V administration of oxytocin to rats can both prevent and treat diet-induced obesity. PMID:26791828

  20. Exosomes Derived From Natural Killer Cells Exert Therapeutic Effect in Melanoma

    PubMed Central

    Zhu, Liya; Kalimuthu, Senthilkumar; Gangadaran, Prakash; Oh, Ji Min; Lee, Ho Won; Baek, Se Hwan; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

    2017-01-01

    Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells. However, the characteristics and functions of exosomes derived from NK cells remain unknown. In this study, we explored NK cell-derived exosome-mediated antitumor effects against aggressive melanoma in vitro and in vivo. Methods: B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads. The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays. Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation. NK-92 Exo were characterized by transmission electron microscopy and western blotting. We also performed an enzyme-linked immunosorbent assay to measure cytokines retained in NK-92 Exo cells. The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays. To investigate the possible side effects of NK-92 Exo on healthy cells, we also performed the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line. Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells. In vivo, we used a B16F10/effluc cell xenograft model to detect the immunotherapeutic effect of NK-92 Exo. We injected NK-92 Exo into tumors, and tumor growth progression was monitored using the IVIS Lumina

  1. Three-phase bone scan and indium white blood cell scintigraphy following porous coated hip arthroplasty: A prospective study of the prosthetic tip

    SciTech Connect

    Oswald, S.G.; Van Nostrand, D.; Savory, C.G.; Callaghan, J.J. )

    1989-08-01

    Although few reports address the use of three-phase bone scanning (TPBS) and {sup 111}In-labeled white blood cell (In-WBC) scintigraphy in hip arthroplasty utilizing a porous coated prosthesis, the literature suggests that scintigraphic patterns in the uncomplicated patient may differ from that seen in the cemented prosthesis. In an attempt to determine the scintigraphic natural history, 25 uncomplicated porous coated hip arthroplasties in 21 patients were prospectively studied with serial TPBS and In-WBC at approximately 7 days, and at 3, 6, 12, 18, and 24 mo postoperatively. This report deals with findings related to the prosthetic tip. Only one of 136 flow studies were abnormal and only two of 136 blood-pool images demonstrated focally increased activity. All 25 prostheses (120 of 143 scans) demonstrated increased uptake on the bone phase images. The area about the tip was divided into three segments; increased uptake at 24 mo was noted in the medial, distal, and lateral segments in 16%, 72%, and 56% of prostheses, respectively. Twenty of 25 prostheses (82 of 142 scans) showed uptake on In-WBC scintigraphy, being noted in 48% of prostheses at 24 mo. We conclude that scintigraphic patterns in the uncomplicated patient with a porous coated prosthesis appear to differ from patterns described in cemented prostheses.

  2. Scintigraphic detection of bone and joint infections with indium-111-labeled nonspecific polyclonal human immunoglobulin G

    SciTech Connect

    Oyen, W.J.; Claessens, R.A.; van Horn, J.R.; van der Meer, J.W.; Corstens, F.H. )

    1990-04-01

    The utility of indium-111-({sup 111}In) labeled immunoglobulin G (IgG) to detect infection of bone and adjacent tissues was investigated. Proof of infection was obtained by cultures taken at surgery. All 32 patients showed focally increased uptake on the technetium-99m- (99mTc) methylene diphosphonate (MDP) skeletal scintigraphies. Labeled immunoglobulin correctly identified presence, location, extent and soft-tissue involvement of the suspected inflammatory site. In these patients, focally increasing accumulation was noted over 48 hr. Discrimination between infection and sterile inflammatory lesions was not possible. Two fractures, 6-mo-old, and an aseptic loosening of a total-hip prosthesis were not visualized. Side effects after the immunoglobulin administration were not observed. Radiolabeled immunoglobulin is a new and safe radiopharmaceutical for the investigation of infectious bone and joint disease. The sensitivity of this agent appears at least as high as that of labeled leukocytes. However, labeled immunoglobulin can easily be prepared in every nuclear medicine department.

  3. Synthesis, pharmacokinetics, and biological use of lysine-modified single-walled carbon nanotubes.

    PubMed

    Mulvey, J Justin; Feinberg, Evan N; Alidori, Simone; McDevitt, Michael R; Heller, Daniel A; Scheinberg, David A

    2014-01-01

    We aimed to create a more robust and more accessible standard for amine-modifying single-walled carbon nanotubes (SWCNTs). A 1,3-cycloaddition was developed using an azomethine ylide, generated by reacting paraformaldehyde and a side-chain-Boc (tert-Butyloxycarbonyl)-protected, lysine-derived alpha-amino acid, H-Lys(Boc)-OH, with purified SWCNT or C60. This cycloaddition and its lysine adduct provides the benefits of dense, covalent modification, ease of purification, commercial availability of reagents, and pH-dependent solubility of the product. Subsequently, SWCNTs functionalized with lysine amine handles were covalently conjugated to a radiometalated chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The (111)In-labeled construct showed rapid renal clearance in a murine model and a favorable biodistribution, permitting utility in biomedical applications. Functionalized SWCNTs strongly wrapped small interfering RNA (siRNA). In the first disclosed deployment of thermophoresis with carbon nanotubes, the lysine-modified tubes showed a desirable, weak SWCNT-albumin binding constant. Thus, lysine-modified nanotubes are a favorable candidate for medicinal work.

  4. The effect of ibuprofen on accumulation of indium-111-labeled platelets and leukocytes in experimental myocardial infarction

    SciTech Connect

    Romson, J.L.; Hook, B.G.; Rigot, V.H.; Schark, M.A.; Swanson, D.P.; Lucchesi, B.R.

    1982-11-01

    To assess the ability of ibuprofen to influence the extent of platelet aggregation and leukocyte infiltration during acute myocardial infarction, autologous indium-111 (/sup 111/In)-labeled platelets or leukocytes were injected before 60 minutes of left circumflex coronary artery (LCx) occlusion, followed by 24 hours of reperfusion in the canine heart. Myocardial infarct size, as a percent of the area at risk, was reduced in the ibuprofen-treated group (12.5 mg/kg i.v. every 4 hours beginning 30 minutes before LCx occulsion) by 40%, from 48 +/- 4% in control animals to 29 +/- 4% in ibuprofen-treated dogs (p=0.005). Quantification of the platelet-associated /sup 111/In radioactivity in irreversibly injured myocardium indicated that ibuprofen did not alter the accumulation of platelets in infarcted myocardium. In contrast, leukocyte accumulation in infarcted tissue was reduced significantly. In tissue samples with 0.41-0.60 gram infarct, the infarcted/normal ratio of leukocyte radioactivity was 12 +/- 2 in control dogs and 4 +/- 1 in ibuprofen-treated dogs, which represents a 67% reduction in leukocyte accumulation in ibuprofen-treated compared with control dogs. Similar reductions were found in other gram-infarct-weight categories. Although both platelets and leukocytes acumulate in infarcted canine myocardium, ibuprofen may exert its beneficial effect on ischemic myocardium by suppressing the inflammatory response associated with myocardial ischemia and infarction.

  5. Amino acid infusion blocks renal tubular uptake of an indium-labelled somatostatin analogue.

    PubMed Central

    Hammond, P. J.; Wade, A. F.; Gwilliam, M. E.; Peters, A. M.; Myers, M. J.; Gilbey, S. G.; Bloom, S. R.; Calam, J.

    1993-01-01

    The Indium-labelled somatostatin analogue pentetreotide has been successfully developed for imaging of somatostatin receptor positive tumours. However there is significant renal tubular uptake of the radiolabelled peptide, which can obscure upper abdominal tumours and would preclude its use for targeted radiotherapy. The aim of this study was to determine whether amino acid infusion, which has been shown to block renal tubular peptide reabsorption, diminishes renal parenchymal uptake of this radiolabelled analogue. Eight patients being scanned with the 111In-labelled somatostatin analogue, pentetreotide, for localisation of gastroenteropancreatic tumours received an infusion of synthetic amino acids. The ratio of isotope uptake in kidney to that in spleen was assessed, and compared to the ratio for matched control patients, to determine if amino acid infusion reduced renal parenchymal uptake of the radiopharmaceutical. The amount of isotope in the urine was determined to ensure that any effect of the amino acid infusion was unrelated to changes in clearance. Infusion of amino acids significantly reduced renal parenchymal uptake of isotope at 4 h. There was a non-significant increase in urinary clearance of isotope over the 4 h, consistent with reduced reuptake and a lack of effect on glomerular filtration rate. This technique, by preventing renal damage, may allow the use of this somatostatin analogue for local radiotherapy, and could be of wider value in blocking tubular re-uptake of potentially nephrotoxic agents, such as radiolabelled Fab fragments. Images Figure 1 PMID:8099808

  6. Monitoring of cardiac antirejection therapy with /sup 111/In lymphocytes

    SciTech Connect

    Lerch, R.A.; Bergmann, S.R.; Carlson, E.M.; Saffitz, J.E.; Sobel, B.E.

    1982-06-01

    To determine whether lymphocytes labeled with /sup 111/In permit noninvasive assessment of antirejection therapy, we performed 40 allogeneic heterotopic cardiac transplants in rats. Antirejection therapy with azathioprine (30 mg/kg) and sodium salicylate (200 mg/kg) prolonged contractile function of the graft from 7.5 +/- 1.5 (s.d.) days in controls to 19.4 +/- 3.7 days in treated animals. Six to seven days after transplantation, autologous lymphocytes labeled with /sup 111/In were injected intravenously in seven untreated and eight treated rats. Scintigraphy and organ counting were performed 24 hr after administration of labeled cells. At sacrifice all grafts in untreated rats exhibited contractile failure, whereas grafts in all treated rats were beating well. Transplants in untreated recipients exhibited marked accumulation of /sup 111/In lymphocytes detectable scintigraphically, with ratios of 7.7 +/- 1.9 for the activity in the transplant over that in the native heart (HT/HO), as obtained by well counting. In contrast, accumulation was not scintigraphically detectable in transplants of treated rats, with HT/HO ratios of 2.6 +/- 1.8 (p less than 0.005). The results suggested that imaging with /sup 111/In-labeled lymphocytes will permit noninvasive assessment of antirejection therapy.

  7. In vivo integrity of polymer-coated gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Kreyling, Wolfgang G.; Abdelmonem, Abuelmagd M.; Ali, Zulqurnain; Alves, Frauke; Geiser, Marianne; Haberl, Nadine; Hartmann, Raimo; Hirn, Stephanie; de Aberasturi, Dorleta Jimenez; Kantner, Karsten; Khadem-Saba, Gülnaz; Montenegro, Jose-Maria; Rejman, Joanna; Rojo, Teofilo; de Larramendi, Idoia Ruiz; Ufartes, Roser; Wenk, Alexander; Parak, Wolfgang J.

    2015-07-01

    Inorganic nanoparticles are frequently engineered with an organic surface coating to improve their physicochemical properties, and it is well known that their colloidal properties may change upon internalization by cells. While the stability of such nanoparticles is typically assayed in simple in vitro tests, their stability in a mammalian organism remains unknown. Here, we show that firmly grafted polymer shells around gold nanoparticles may degrade when injected into rats. We synthesized monodisperse radioactively labelled gold nanoparticles (198Au) and engineered an 111In-labelled polymer shell around them. Upon intravenous injection into rats, quantitative biodistribution analyses performed independently for 198Au and 111In showed partial removal of the polymer shell in vivo. While 198Au accumulates mostly in the liver, part of the 111In shows a non-particulate biodistribution similar to intravenous injection of chelated 111In. Further in vitro studies suggest that degradation of the polymer shell is caused by proteolytic enzymes in the liver. Our results show that even nanoparticles with high colloidal stability can change their physicochemical properties in vivo.

  8. In vivo integrity of polymer-coated gold nanoparticles.

    PubMed

    Kreyling, Wolfgang G; Abdelmonem, Abuelmagd M; Ali, Zulqurnain; Alves, Frauke; Geiser, Marianne; Haberl, Nadine; Hartmann, Raimo; Hirn, Stephanie; de Aberasturi, Dorleta Jimenez; Kantner, Karsten; Khadem-Saba, Gülnaz; Montenegro, Jose-Maria; Rejman, Joanna; Rojo, Teofilo; de Larramendi, Idoia Ruiz; Ufartes, Roser; Wenk, Alexander; Parak, Wolfgang J

    2015-07-01

    Inorganic nanoparticles are frequently engineered with an organic surface coating to improve their physicochemical properties, and it is well known that their colloidal properties may change upon internalization by cells. While the stability of such nanoparticles is typically assayed in simple in vitro tests, their stability in a mammalian organism remains unknown. Here, we show that firmly grafted polymer shells around gold nanoparticles may degrade when injected into rats. We synthesized monodisperse radioactively labelled gold nanoparticles ((198)Au) and engineered an (111)In-labelled polymer shell around them. Upon intravenous injection into rats, quantitative biodistribution analyses performed independently for (198)Au and (111)In showed partial removal of the polymer shell in vivo. While (198)Au accumulates mostly in the liver, part of the (111)In shows a non-particulate biodistribution similar to intravenous injection of chelated (111)In. Further in vitro studies suggest that degradation of the polymer shell is caused by proteolytic enzymes in the liver. Our results show that even nanoparticles with high colloidal stability can change their physicochemical properties in vivo.

  9. Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

    PubMed Central

    Smith, Andrew M.; Rahman, Farooq Z.; Hayee, Bu'Hussain; Graham, Simon J.; Marks, Daniel J.B.; Sewell, Gavin W.; Palmer, Christine D.; Wilde, Jonathan; Foxwell, Brian M.J.; Gloger, Israel S.; Sweeting, Trevor; Marsh, Mark; Walker, Ann P.; Bloom, Stuart L.

    2009-01-01

    The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of 111In-labeled neutrophils at these sites and clearance of 32P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway. PMID:19652016

  10. Convenient solid-phase synthesis of diethylenetriaminepenta-acetic acid (DTPA)- conjugated cyclic RGD peptide analogues.

    PubMed

    Wang, Wei; McMurray, John S; Wu, Qingping; Campbell, Martin L; Li, Chun

    2005-10-01

    Solid-phase synthesis of radiometal chelator-conjugated peptides can facilitate the creation of radioactive peptide libraries to be utilized in high throughput in vivo screening of targeted nuclear-imaging agents. In this study, a new diethylenetriaminepentaacetic acid (DTPA) derivative, 1-(p-succinamidobenzyl)- DTPA penta-t-butyl ester [DTPA(But)(5)-Bz-NH-SA], and its precursor molecule, 1-(p-aminobenzyl)- DTPA penta-t-butyl ester (DTPA(But)(5)-Bz-NH(2)), were applied to the solid-phase synthesis of DTPA-conjugated cyclic peptides containing the Arg-Gly-Asp (RGD) motif with high efficiency. The resulting conjugates, DTPA-Bz-NH-SA-c(Lys-Arg-Gly-Asp-phe) [DTPA-Bz-NH-SA-c(KRGDf)] and DTPA-Bz-NHc( Glu-Arg-Gly-Asp-phe) [DTPA-Bz-NH-c(KRGDf)], demonstrated similar in vitro biologic activities as their corresponding parent peptides. (111)In-labeled, DTPA-conjugated RGD peptides showed selective binding to integrin alphavbeta3 in human melanoma M21 tumors grown in nude mice. Furthermore, (111)In-DTPABz- NH-c(ERGDf) showed lower retention in the liver and the kidney than (111)In-DTPA-Bz-NH-SAc( KRGDf) did, which contributed to higher target to nontarget ratio for (111)In-DTPA-Bz-NH-c(ERGDf). The method reported here can be extended to the construction of peptide libraries containing DTPA for high throughput in vitro and in vivo screening of molecularly targeted imaging agents.

  11. In vivo Stability of Ester- and Ether-Linked Phospholipid-Containing Liposomes as Measured by Perturbed Angular Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Derksen, Johannes T.; Baldeschwieler, John D.; Scherphof, Gerrit L.

    1988-12-01

    To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing 111In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of 111In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor 111In of the [G22(∞ )] label. We found that MLVs but not SUVs, having DHPC as their bulk phospholipid, showed an increased resistance against lysosomal degradation as compared to other phospholipid-containing liposomes. The use of diacyl phospholipids with a high gel/liquid-crystalline phase-transition temperature, such as DPPC and DSPC, also retarded degradation of MLV, but not of SUV in the dose range tested, while the rate of uptake of these liposomes by the liver was lower.

  12. Model of rapid gastrointestinal transit in dogs: effects of muscarinic antagonists and a nitric oxide synthase inhibitor.

    PubMed

    Chiba, T; Bharucha, A E; Thomforde, G M; Kost, L J; Phillips, S F

    2002-10-01

    Our aims were to establish a canine model of rapid gastrointestinal transit, and to test the effects of muscarinic receptor antagonists (atropine, pirenzepine, AF-DX116, and darifenacin), and an NOS inhibitor, L-nitro-N-arginine (L-NNA) in this model. For gastric emptying and small bowel transit, 99mTc-labelled DTPA were added to a meal of skimmed milk (236 mL) that contained 2.4 g of magnesium hydroxide. Regional colonic transit was measured by111In-labelled beads placed in a capsule that released isotope in the proximal colon. Scintiscans were taken at regular intervals and indices of transit were calculated. Drugs were administrated intravenously. Gastric emptying, small bowel and colonic transit were rapid. Atropine and darifenacin (a selective M3 antagonist) delayed gastric emptying and colonic transit, the selective M1 and M2 muscarinic antagonists did not. The muscarinic blockers did not slow small bowel transit. L-NNA delayed small bowel and colonic transit but did not slow gastric emptying. A model suitable for the preclinical study of antidiarrhoeals was established. M3 receptors are important in the control of gastric emptying and colonic transit, and NOS inhibition slowed small bowel and colonic transit.

  13. Pretargeted immunoscintigraphy in patients with medullary thyroid carcinoma.

    PubMed Central

    Magnani, P.; Paganelli, G.; Songini, C.; Samuel, A.; Sudati, F.; Siccardi, A. G.; Fazio, F.

    1996-01-01

    To evaluate the use of pretargeted immunoscintigraphy (ISG) in the diagnosis and follow-up of patients with medullary thyroid carcinoma (MTC), we studied 25 patients with histologically proven disease; ISG was repeated after surgery in two patients. The antibody, either an anticarcinoembryonic antigen (CEA) or an antichromogranin A (CgA) biotinylated monoclonal antibody (MAb) or a cocktail of the two biotinylated MAbs was first injected. After 24 h, avidin was administrated i.v., followed by 111In-labelled biotin 24 h later. Fifty-two lesions were visualised. Six primary tumours, diagnosed by increased calcitonin levels, were all correctly diagnosed; 47 recurrences, also suspected by blood tumour markers, were detected and confirmed by cytology or histology. In one case, single photon emission tomography allowed the detection of small lymph nodes with a diameter of 4-7 mm. These lesions, not judged neoplastic by ultrasound, were confirmed to be neoplastic by fine needle aspiration. Pretargeted ISG correctly localises primary tumours and recurrences in MTC patients, when the only marker of relapse is serum elevation of calcitonin. With this three-step pretargeting method, cocktails of potentially useful MAbs can be used, avoiding false-negative studies that may occur when CEA or CgA are not expressed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8795589

  14. Histogram analysis of pharmacokinetic parameters by bootstrap resampling from one-point sampling data in animal experiments.

    PubMed

    Takemoto, Seiji; Yamaoka, Kiyoshi; Nishikawa, Makiya; Takakura, Yoshinobu

    2006-12-01

    A bootstrap method is proposed for assessing statistical histograms of pharmacokinetic parameters (AUC, MRT, CL and V(ss)) from one-point sampling data in animal experiments. A computer program, MOMENT(BS), written in Visual Basic on Microsoft Excel, was developed for the bootstrap calculation and the construction of histograms. MOMENT(BS) was applied to one-point sampling data of the blood concentration of three physiologically active proteins ((111)In labeled Hsp70, Suc(20)-BSA and Suc(40)-BSA) administered in different doses to mice. The histograms of AUC, MRT, CL and V(ss) were close to a normal (Gaussian) distribution with the bootstrap resampling number (200), or more, considering the skewness and kurtosis of the histograms. A good agreement of means and SD was obtained between the bootstrap and Bailer's approaches. The hypothesis test based on the normal distribution clearly demonstrated that the disposition of (111)In-Hsp70 and Suc(20)-BSA was almost independent of dose, whereas that of (111)In-Suc(40)-BSA was definitely dose-dependent. In conclusion, the bootstrap method was found to be an efficient method for assessing the histogram of pharmacokinetic parameters of blood or tissue disposition data by one-point sampling.

  15. Faecal alpha-1-antitrypsin and excretion of 111indium granulocytes in assessment of disease activity in chronic inflammatory bowel diseases.

    PubMed Central

    Fischbach, W; Becker, W; Mössner, J; Koch, W; Reiners, C

    1987-01-01

    Intestinal protein loss in chronic inflammatory bowel diseases may be easily determined by measurement of alpha-1-antitrypsin (alpha 1-AT) stool concentration and alpha 1-AT clearance. Both parameters were significantly raised in 36 and 34 patients respectively with chronic inflammatory bowel diseases, compared with eight patients with non-inflammatory bowel diseases, or 19 healthy volunteers. There was wide range of overlap between active and inactive inflammatory disease. Contrary to serum alpha 1-AT, faecal excretion and clearance of alpha 1-AT did not correlate with ESR, serum-albumin, orosomucoid, and two indices of disease activity. A comparison of alpha 1-AT faecal excretion and clearance with the faecal excretion of 111In labelled granulocytes in 27 patients with chronic inflammatory bowel diseases, showed no correlation between the intestinal protein loss and this highly specific marker of intestinal inflammation. Enteric protein loss expressed by faecal excretion and clearance of alpha 1-AT does not depend on mucosal inflammation only, but may be influenced by other factors. PMID:3495470

  16. Dual isotope study of iodine-125 and indium-111-labeled antibody in athymic mice

    SciTech Connect

    Carney, P.L.; Rogers, P.E.; Johnson, D.K. )

    1989-03-01

    Monoclonal antibody B72.3 was coupled to a benzylisothiocyanate derivative of diethylenetriaminepentaacetic acid (DTPA). The maximum substitution achievable without loss of immunoreactivity was three DTPA groups per immunoglobulin molecule. The resulting conjugate was labeled with {sup 111}In by brief incubation with {sup 111}InCl{sub 3}, giving a mean radiochemical yield of {sup 111}In-labeled antibody of 96%. The ({sup 111}In)B72.3 preparation was mixed with an ({sup 125}I) B72.3 preparation, obtained by the chloramine-T method, and the mixture administered to athymic mice bearing subcutaneous LS174T colon carcinoma xenografts. There were no significant differences (p greater than 0.1) in the biodistributions of the two labels at 1, 2, 5, and 7 days postinjection. These results are contrasted with prior studies showing elevated levels of {sup 111}In in liver, spleen, and kidneys using B72.3-DTPA conjugates prepared via the bicyclic anhydride. It is concluded that protein cross-linking and/or the formation of unstable chelate sites in anhydride coupled conjugates underlie these disparities.

  17. Modulation of acute immune complex-mediated tissue injury by the presence of polyionic substances.

    PubMed Central

    Warren, J. S.; Ward, P. A.; Johnson, K. J.; Ginsburg, I.

    1987-01-01

    Considerable attention has been focused on the role of electrostatic charge in the pathogenesis of immune complex-mediated tissue injury. The authors have examined the ability of cationic (histone, polyhistidine, polyarginine) and anionic (polyanetholsulfonate) polyelectrolytes to modulate acute immune complex-mediated tissue injury. Tissue injury elicited in rats by the reversed dermal Arthus reaction was increased 26-43% by addition of polyelectrolytes to antibody prior to its intradermal injection. Kinetic studies using 111In-labeled neutrophils indicated that the enhanced tissue injury was not the result of increased influx of neutrophils. Infusion of polyethylene glycol-conjugated superoxide dismutase prior to induction of the Arthus reaction resulted in 40-68% suppression of tissue injury. Concomitant in vitro functional studies (enzyme secretion, O-2 and H2O2 generation, and chemiluminescence) of rat neutrophils demonstrated that addition of polyelectrolytes to preformed immune complexes (IgG-bovine serum albumin) resulted in marked increases in O-2, H2O2, and chemiluminescence, but no increases in enzyme secretion, compared with neutrophils stimulated with immune complexes alone. The cationic polyelectrolytes did not alter the capacity of preformed immune complexes to activate complement in vitro. These studies suggest that both cationic and anionic polyelectrolytes can increase the pathogenic potential of immune complexes and that this modulation is, at least in part, mediated by enhanced generation of toxic oxygen-derived metabolites by neutrophils. PMID:3037912

  18. Role of indium-111 white blood cells in inflammatory bowel disease

    SciTech Connect

    Froelich, J.W.; Field, S.A.

    1988-10-01

    Inflammatory bowel disease in patients may be difficult to diagnose because of the complex problems associated with this disease. Radionuclides are able to provide a rapid and effective method of imaging the bowel in patients with active inflammatory bowel disease. In the past, clinical work-ups have included barium x-ray studies and endoscopy. Scarring and fistula formation have made it difficult to determine between the active disease and abscesses that may occur. Gallium-67 (67Ga) has been very useful in imaging patients with inflammatory bowel disease, but the multiple-day imaging procedure has been a limitation for the clinicians when achieving a diagnosis. Recent results with Indium-111 (111In)--labeled WBCs have provided excellent correlation between clinical symptoms and colonoscopy findings in patients with inflammatory bowel disease. This technique has also allowed the differentiation between reoccurring inflammatory bowel disease and abscesses that accompany the disease within a 24-hour time period. The use of intravenous (IV) glucagon has increased the clarity of the images in the small bowel. Technetium 99m (99mTc) diethylenetriaminepentaacetic acid (DTPA) has been used in patients with inflammatory bowel disease demonstrating promising results. Investigators feel labelling 99mTc with WBCs will be improved, therefore yielding a greater efficiency, which will have a major impact on imaging patients with inflammatory bowel disease. Imaging patients with inflammatory bowel disease using radionuclides has yielded promising results. This is a significant advancement over barium radiography and endoscopy exams.24 references.

  19. Effects of radiolabelled monoclonal antibody infusion on blood leukocytes in cancer patients

    SciTech Connect

    Gridley, D.S.; Slater, J.M.; Stickney, D.R. )

    1990-01-01

    This study was undertaken to investigate the effects of a single infusion of radiolabelled murine monoclonal antibody (MAb) on peripheral blood leukocytes in cancer patients. Eleven patients with disseminated colon cancer, malignant melanoma, or lung adenocarcinoma were infused with 111In-labelled anti-ZCE 025, anti-p97 type 96.5c, or LA 20207 MAb, respectively. Blood samples were obtained before infusion, immediately after infusion (1 hr), and at 4 and 7 days postinfusion. Flow cytometry analysis of CD3+, CD4+, CD8+, CD16+, and CD19+ lymphocytes showed increasing CD4:CD8 ratios in seven patients after infusion. This phenomenon was not restricted to antibody subclass or to type of cancer. Two of the remaining patients exhibited a marked post-infusion increase in CD8+ cells. In all three patients with malignant melanoma, decreasing levels of CD16+ lymphocytes were noted after infusion and natural killer cell cytotoxicity showed fluctuations which paralleled the changes in the CD16+ subpopulation. Oxygen radical production by phagocytic cells was markedly affected in three subjects. These results suggest that a single infusion of radiolabelled murine MAb may alter the balance of critical lymphocyte subpopulations and modulate other leukocyte responses in cancer patients.

  20. Gold nanorod-mediated hyperthermia enhances the efficacy of HPMA copolymer - 90Y conjugates in treatment of prostate tumors

    PubMed Central

    Buckway, Brandon; Frazier, Nick; Gormley, Adam J.; Ray, Abhijit; Ghandehari, Hamidreza

    2014-01-01

    Introduction The treatment of prostate cancer using a radiotherapeutic 90Y labeled N-(2-hydroxypropyl)methacrylamide (HPMA)copolymer can be enhanced with localized tumor hyperthermia. An 111In labeled HPMA copolymer system for single photon emission computerized tomography (SPECT) was developed to observe the biodistribution changes associated with hyperthermia. Efficacy studies were conducted in prostate tumor bearing mice using the 90Y HPMA copolymer with hyperthermia. Methods HPMA copolymers containing 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were synthesized by reversible addition-fragmentation transfer (RAFT) copolymerization and subsequently labeled with either 111In for imaging or 90Y for efficacy studies. Radiolabel stability was characterized in vitro with mouse serum. Imaging and efficacy studies were conducted in DU145 prostate tumor bearing mice. Imaging was performed using single photon emission computerized tomography (SPECT). Localized mild tumor hyperthermia was achieved by plasmonic photothermal therapy using gold nanorods. Results HPMA copolymer-DOTA conjugates demonstrated efficient labeling and stability for both radionuclides. Imaging analysis showed a marked increase of radiolabeled copolymer within the hyperthermia treated prostate tumors, with no significant accumulation in non-targeted tissues. The greatest reduction in tumor growth was observed in the hyperthermia treated tumors with 90Y HPMA copolymer conjugates. Histological analysis confirmed treatment efficacy and safety. Conclusion HPMA copolymer-DOTA conjugates radiolabeled with both the imaging and treatment radioisotopes, when combined with hyperthermia can serve as an image guided approach for efficacious treatment of prostate tumors. PMID:24461626

  1. Imaging Lung Clearance of Radiolabeled Tumor Cells to Study Mice with Normal, Activated or Depleted Natural Killer (NK) Cells

    NASA Astrophysics Data System (ADS)

    Kulkarni, P. V.; Bennett, M.; Constantinescu, A.; Arora, V.; Viguet, M.; Antich, P.; Parkey, R. W.; Mathews, D.; Mason, R. P.; Oz, O. K.

    2003-08-01

    Lung clearance of 51CR and 125I iododeoxyuridine (IUDR) labeled cancer cells assess NK cell activity. It is desirable to develop noninvasive imaging technique to assess NK activity in mice. We labeled target YAC-1 tumor cells with 125I, 111In, 99mTc, or 67Ga and injected I.V. into three groups of BALB/c mice. Animals were treated with medium (group I), 300mg/kg cyclophosmamide (CY) to kill NK cell (group II), or anti-LY49C/1) (ab')2 mAb to augment NK function (group III). Lungs were removed 15 min or 2 h later for tissue counting. Control and treated mice were imaged every 5 min with a scintillating camera for 1 h after 15 min of infusion of the 111In labeled cells. Lung clearance increased after 15 min (lodging: 60-80%) and (2 h retention: 3-7%). Similar results were obtained with all the isotopes studied. Images distinguished the control and treated mice for lung activity. Cells labeled with 111In, 99mTc or 67Ga are cleared similar to those labeled with 51Cr or 125I. NK cell destruction of tumor cells may be assessed by noninvasive imaging method either by SPECT (99mTc, 111In, 67Ga) or by PET (68Ga).

  2. Ultraviolet irradiation of platelet concentrates: Feasibility in transfusion practice

    SciTech Connect

    Andreu, G.; Boccaccio, C.; Lecrubier, C.; Fretault, J.; Coursaget, J.; LeGuen, J.P.; Oleggini, M.; Fournel, J.J.; Samama, M. )

    1990-06-01

    Ultraviolet (UV)-B irradiation abolishes lymphocyte functions (the ability to respond and to stimulate) in mixed lymphocyte culture (MLC). This effect may have practical application in the prevention or reduction of transfusion-induced alloimmunization against HLA class I antigens. To study this, platelet concentrates (PCs) were obtained with a cell separator, suspended in autologous plasma in a final volume of 400 mL, and transferred into a large (22 X 30 cm) cell culture bag. This plastic showed a good transmittance of UV-B rays at 310 nm (54%). PCs were placed between two quartz plates (surface of irradiation = 25 X 37 cm), and the two sides were irradiated simultaneously. Energy delivered to the surface of the plastic bag was automatically monitored. The ability to respond (in MLC and to phytohemagglutinin) and to stimulate allogeneic lymphocytes was completely abolished with energy of 0.75 J per cm2 (irradiation time less than 3 min). The temperature increase during irradiation was negligible. Platelet aggregation (collagen, adrenalin, ADP, arachidonic acid, ristocetin) was not impaired if UV-B energy was below 3 J per cm2. Recovery and survival of autologous 111In-labeled platelets were studied in four volunteers; no differences were found between UV-B-treated (1.5 J/cm2) platelets and untreated platelets. These results show that a large-scale clinical trial using UV-B-irradiated PCs to prevent HLA alloimmunization is feasible.

  3. Imaging the inflammatory response to acute myocardial infarction in man using indium-111-labeled autologous platelets

    SciTech Connect

    Davies, R.A.; Thakur, M.L.; Berger, H.J.; Wackers, F.J.T.; Gottschalk, A.; Zaret, B.L.

    1981-04-01

    The feasibility of imaging the inflammatory response to acute transmural myocardial infarction in man using indium-111 (/sup 111/In)-labeled autologous leukocytes was assessed in 36 patients. Indium-111 leukocytes were injected i.v. 18 to 112 hs after the onset of chest pain. Cardiac imaging was performed 24 hs later with a mobile gamma camera. Twenty-one patients had positive images and 15 had negative images. The percent of positive images increased as the interval between infarction and /sup 111/In-leukocyte injection shortened; all patients injected within 24 hs of infarction had positive images. Patients with positive images were injected with /sup 111/In leukocytes earlier after infarction and were younger than those with negative images. Several other parameters that could possibly have affected the imaging results were examined and were not significantly different in patients with positive and negative images. These included peak serum creatine kinase, location of infarction, incidence of pericarditis, use of antiinflammatory drugs or membrane-active antiarrhythmic drugs, peripheral leukocyte count, and cell labeling efficiency. The function of the labeled cells was similar in patients with positive and negative images. Six patients with acute infarction serving as controls and given free /sup 111/In-oxine and six patients with stable coronary artery disease given /sup 111/In-leukocytes all had negative cardiac images.

  4. Mechanisms and kinetics for platelet and neutrophil localization in immune complex nephritis

    SciTech Connect

    Johnson, R.J.; Alpers, C.E.; Pruchno, C.; Schulze, M.; Baker, P.J.; Pritzl, P.; Couser, W.G. )

    1989-11-01

    We have previously reported that both neutrophils (PMNs) and platelets mediate proteinuria in a model of subendothelial immune complex (IC) nephritis (GN) in the rat. In order to understand the interaction of PMNs and platelets in this model, we quantitated the uptake of {sup 111}In-labelled platelets in glomeruli and correlated this with the number of PMNs observed histologically at 10 and 30 minutes, 1, 4 and 24 hours following induction of GN. Platelet accumulation was biphasic with a major peak at 10 minutes and a minor peak at four hours. Early platelet accumulation was complement dependent, and PMN-independent. PMN accumulation occurred after the initial platelet influx, peaking at one and four hours, was complement dependent, but was not affected by platelet depletion. Complement depletion significantly reduced proteinuria. This is the first documentation that platelet accumulation in glomeruli in IC GN is complement dependent. In addition, the enhancement of PMN-mediated injury by the platelet in this model does not involve effects of platelets on PMN localization, thus implying a functional interaction between these cells within the glomerulus.

  5. Splenic microenvironment and self recognition as factors in allograft rejection in rats. A study using indium-111-labeled cells

    SciTech Connect

    Pollak, R.; Blanchard, J.M.; Lazda, V.A.

    1986-11-01

    Splenectomy facilitates organ allograft survival in some rat strains, and in weak donor-recipient histoincompatible pairs. We have found using a heart spleen twin graft model, using ACI rats as recipients and Lewis rats as donors, that the transplanted heart will survive in most recipients after delayed host splenectomy. The presence of a viable mass of splenic tissue will allow rejection to proceed only when the transplanted spleen is of host origin, and not when it comes from the donor (i.e., when it is allogeneic). The use of 111In-labeled cells has allowed us to show that lymphocyte traffic and trapping is markedly altered in the transplanted allogeneic spleens, when compared with control transplanted syngeneic spleens. Thus, despite the presence of the splenic ''microenvironment,'' cardiac allograft rejection does not occur in the absence of syngeneic splenic tissue. We conclude that the role of the spleen in the immune response is to facilitate the recognition of self and the acquisition of alloreactivity in weak responder rat strains and donor-recipient pairs.

  6. Evaluation of the viability of /sup 111/In-abeled DTPA coupled to fibrinogen

    SciTech Connect

    Layne, W.W.; Hnatowich, D.J.; Doherty, P.W.; Childs, R.L.; Lanteigne, D.; Ansell, J.

    1982-07-01

    In earlier work, DTPA has been covalently coupled to albumin via the cyclic anhydride of DTPA. Using fibrinogen, we have studied the effect of such coupling on protein viability by both an in vitro and an in vivo assay. Clotting time remained identical to that of the native protein whether the anhydride-to-protein molar ratio was 1:1 or 5:1. In vivo studies were done in dogs, with human fibrinogen labeled with /sup 125/I and /sup 111/In. Throughout 130 hr, blood clearances for the two tracers agreed whether with 1:1 or 5:1 coupling. In a dog model with a thrombogenic catheter, the clot-to-blood ratios for the two radiotracers agreed within experimental error. Finally, 1:1-coupled canine fibrinogen, labeled with /sup 111/In, was administered to dogs with a catheter in a jugular vein, and scintigrams at 24 hr clearly showed clotting along the length of the catheter. We conclude that fibrinogen, coupled to DTPA, retains its viability, behaving like radioiodinated fibrinogen in vivo, and /sup 111/In labeled fibrinogen looks promising as a clinical diagnostic agent.

  7. Indium-111-labeled LDL: A potential agent for imaging atherosclerotic disease and lipoprotein biodistribution

    SciTech Connect

    Rosen, J.M.; Butler, S.P.; Meinken, G.E.; Wang, T.S.; Ramakrishnan, R.; Srivastava, S.C.; Alderson, P.O.; Ginsberg, H.N. )

    1990-03-01

    Radiolabeling of low-density lipoprotein (LDL) and external imaging with a gamma camera would offer a means of taking advantage of the metabolic activity of developing atherosclerotic lesions in order to noninvasively detect and determine the extent of atherosclerotic cardiovascular disease. Indium-111-({sup 111}In) labeled LDL was prepared and its purity demonstrated by agarose electrophoresis and ultracentrifugation. In vitro studies with cultured human fibroblasts demonstrated significant inhibition of iodine-125-({sup 125}I) LDL binding to LDL receptors by {sup 111}In-LDL, although this was less than the inhibition produced by unlabeled LDL. Adrenal gland uptake of {sup 111}In-LDL by hypercholesterolemic rabbits was reduced by 86% compared to the level of uptake observed in normal rabbits. These results were compatible with downregulation of adrenal LDL receptors in the hypercholesterolemic rabbits. Uptake of {sup 111}In-LDL in the atherosclerotic proximal aorta of hypercholesterolemic rabbits was 2.5 times higher than in normal rabbits. These results suggest that {sup 111}In-LDL has the potential to be a useful agent for external imaging of atherosclerotic lesions and lipoprotein biodistribution.

  8. Tumor immunoscintigraphy by means of radiolabeled monoclonal antibodies: Multicenter studies of the Italian National Research Council--Special Project Biomedical Engineering

    SciTech Connect

    Siccardi, A.G. )

    1990-02-01

    Four radioimmunopharmaceuticals ({sup 99m}Tc- and 111In-labeled anti-melanoma and {sup 111}In- and {sup 131}I-labeled anti-carcinoembryonic antigen F(ab')2 fragments derived from monoclonal antibodies 225.28S and F023C5) were developed by means of a collaborative effort coordinated by the Italian National Research Council, Special Project Biomedical Engineering. After appropriate pilot studies, the radioimmunopharmaceuticals, prepared by Sorin Biomedica (Saluggia, Italy), were distributed to 31 Nuclear Medicine departments in Italy and in 10 other European countries within the framework of three immunoscintigraphy multicenter studies. A total of 1245 patients were studied, 898 of whom carried 1725 documented tumor lesions; 1596 of 2193 tumor lesions (468 of which were previously unknown) were imaged by immunoscintigraphy in 785 of 990 lesion-bearing patients. Among the occult lesions, 173 were imaged in 92 patients admitted to the study as lesion-free patients. The results have been analyzed in terms of the reliability, reproducibility, and diagnostic usefulness of the method and of each immunoradiopharmaceutical.

  9. Dose rate measurements from radiopharmaceuticals: implications for nuclear medicine staff and for children with radioactive parents.

    PubMed

    Greaves, C D; Tindale, W B

    1999-02-01

    Following the introduction of a number of radiopharmaceuticals, we assessed the dose received by staff working in the nuclear medicine department and also by children who may be in close contact with a radioactive parent. We measured departure dose rates (microSv.h-1) at distances of 0.1, 0.5 and 1.0 m from the skin surface at the level of the thyroid, chest and bladder of patients undergoing the following nuclear medicine procedures: MUGA scans using 99Tcm-labelled red blood cells, myocardial perfusion scans using 99Tcm-labelled radiopharmaceuticals, lymphoscintigraphy using colloidal 99Tcm (Re) sulphide, bone scans using 99Tcm-labelled oxidronate, 111In-octreotide scans, 111In-labelled leukocyte studies and cardiac reinjection studies using 201Tl. The maximum dose rates at 0.1 m were those from MUGA studies (167.3 microSv.h-1) and myocardial perfusion studies (one-day protocol = 391.7 microSv.h-1, two-day protocol = 121.8 microSv.h-1). The implications of these dose rates on both technical and nursing staff are assessed. Also, the dose received by an infant in close contact with a parent following a nuclear medicine investigation was estimated.

  10. Evaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]-Z HER2:2395(-Cys) affibody molecule for targeting of HER2-expressing tumors.

    PubMed

    Wållberg, Helena; Ahlgren, Sara; Widström, Charles; Orlova, Anna

    2010-01-01

    Imaging using positron emission tomography (PET) in the field of nuclear medicine is becoming increasingly important. The aim of this study was to develop a method for labeling of affibody molecules with radiocobalt for PET applications. The human epidermal growth factor receptors type 2 (HER2) binding affibody molecule DOTA-Z(2395)-C was radiolabeled with (57)Co (used as a surrogate of (55)Co). The binding specificity and cellular processing of the labeled compound was studied in vitro followed by in vivo characterization in normal and tumor-bearing mice. Furthermore, a comparative biodistribution study was performed with a (111)In-labeled counterpart. DOTA-Z(2395)-C was successfully labeled with radiocobalt with nearly quantitative yield. The compound displayed good retention on cells over time and high tumor accumulation of radioactivity in animal studies. Imaging studies showed clear visualization of HER2-positive tumors. Furthermore, the radiocobalt label provided better tumor-to-organ ratios than (111)In. Radiocobalt is a promising label for affibody molecules for future PET applications.

  11. Synthesis, pharmacokinetics, and biological use of lysine-modified single-walled carbon nanotubes

    PubMed Central

    Mulvey, J Justin; Feinberg, Evan N; Alidori, Simone; McDevitt, Michael R; Heller, Daniel A; Scheinberg, David A

    2014-01-01

    We aimed to create a more robust and more accessible standard for amine-modifying single-walled carbon nanotubes (SWCNTs). A 1,3-cycloaddition was developed using an azomethine ylide, generated by reacting paraformaldehyde and a side-chain-Boc (tert-Butyloxycarbonyl)-protected, lysine-derived alpha-amino acid, H-Lys(Boc)-OH, with purified SWCNT or C60. This cycloaddition and its lysine adduct provides the benefits of dense, covalent modification, ease of purification, commercial availability of reagents, and pH-dependent solubility of the product. Subsequently, SWCNTs functionalized with lysine amine handles were covalently conjugated to a radiometalated chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The 111In-labeled construct showed rapid renal clearance in a murine model and a favorable biodistribution, permitting utility in biomedical applications. Functionalized SWCNTs strongly wrapped small interfering RNA (siRNA). In the first disclosed deployment of thermophoresis with carbon nanotubes, the lysine-modified tubes showed a desirable, weak SWCNT-albumin binding constant. Thus, lysine-modified nanotubes are a favorable candidate for medicinal work. PMID:25228803

  12. Kinetic study of platelets and fibrinogen in Lassa virus-infected monkeys and early pathologic events in Mopeia virus-infected monkeys.

    PubMed

    Lange, J V; Mitchell, S W; McCormick, J B; Walker, D H; Evatt, B L; Ramsey, R R

    1985-09-01

    The rhesus monkey, an established model of Lassa fever, was used to study hematologic and hemostatic aspects of Lassa fever and whether Mopeia (also known as Mozambique) virus induces any cellular damage in this model. Six days after subcutaneous injection of 10(3.48) plaque forming units (PFU) of Lassa virus (Josiah strain) one group of monkeys received an intravenous injection of 111In-labeled allogeneic platelets and another group received 125I-labeled alogeneic fibrinogen. Lassa virus-infected monkeys developed a severe clinical illness with high viremia and typical pathology. Lassa antigen was found in most tissues using a Lassa nucleocapsid-specific monoclonal antibody. Platelet counts remained within normal limits. Platelet and fibrinogen kinetics were similar in infected and control animals. Hematologic and hemostatic changes indicate that disseminated intravascular coagulation plays no role in this model of Lassa fever. Levels of plasma fibronectin were reduced in Lassa-infected monkeys. Mopeia virus-infected monkeys were normothemic, aviremic, and there was no detection of Mopeia antigen in any tissues using polyclonal or monoclonal antibodies. Mopeia virus was recovered from the spleen of one monkey. Mopeia virus was associated with hepatocellular and renal tubular damage.

  13. Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease.

    PubMed

    Smith, Andrew M; Rahman, Farooq Z; Hayee, Bu'Hussain; Graham, Simon J; Marks, Daniel J B; Sewell, Gavin W; Palmer, Christine D; Wilde, Jonathan; Foxwell, Brian M J; Gloger, Israel S; Sweeting, Trevor; Marsh, Mark; Walker, Ann P; Bloom, Stuart L; Segal, Anthony W

    2009-08-31

    The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at these sites and clearance of (32)P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

  14. 111-Indium labelled autologous leucocytes in diagnosis of inflammatory bowel disease

    SciTech Connect

    Wandall, J.H.; Edeling, C.J.; Jensen, J.T.; Lund, J.O.; Bonnevie, O.; Haxholdt, H.; Jensen, H.C.; Matzen, P.; Myschetsky, P.S.; Nielsen, A.M.

    1984-01-01

    111-Indium labelled leucocytes have been used to visualize inflammatory lesions in ulcerative colitis (CU) and in Crohn's disease (CD). The aim of this study was to compare findings by scintigraphy, radiology and endoscopy. Material: Twelve patients with CU and 15 patients with CD were studied. All patients were non-febrile. Two patients received prednisolone 5 mg/daily, 8 sulphasalazine. Methods: Autologous leucocytes were labelled with 111-In-Oxine and given i.v. Scintigrams were obtained 3 and 24 hrs. p.i. Double contrast x-ray studies were done of the colon and small intestine after 2 and 14 days respectively. Colonscopy with biopsy was done after 4 days. Results: Active lesions were found in 24 and 27 patients. Scintigrams 24 hrs.p.i. did not give and additional information compared with scintigrams 3 hrs.p.i. Intraluminal activity masked the location and extension of lesions after 24 hrs. Excretion in the stool was 2.4-25.8% of administered activity. Compared with scintigraphy a corresponding extension and location was found by colonscopy. In 4 patients x-ray of the colon was normal but scintigraphy and colonscopy showed active inflammation. Conclusion: Scintigraphy after injection of 111-In labelled leucocytes is a atraumatic method for visualization of inflammatory lesions in UC and CD. Furthermore, it appears to be more sensitive than conventional x-ray studies.

  15. The white blood cell scan in orthopedics

    SciTech Connect

    Propst-Proctor, S.L.; Dillingham, M.F.; McDougall, I.R.; Goodwin, D.

    1982-08-01

    A new nuclear scanning technique was found more specific for bone, joint, and soft tissue infections than any previously described scanning technique. The leukocyte scan, whereby a patient's own cells are labeled with a radioactive tagging agent (/sup 111/In oxine), can distinguish an active infectious process from other pain-inducing conditions. Ninety-seven /sup 111/In labeled autologous leukocyte scans were performed in 88 patients. The findings in 17 of 40 patients scanned for possible acute osteomyelitis, six of nine for suspected septic arthritis, and six for possible soft tissue infections, were positive. Subsequent clinical courses verified the infectious nature of these processes in all patients. Patients who had chronic osteomyelitis (14), bony metastases (four patients), heterotopic ossification (three), and degenerative arthritis (two) demonstrated negative findings. Of the seven patients scanned for acute long-bone fractures, one demonstrated positive findings. Nine scans demonstrated positive findings without determined causes. The leukocyte scan is a useful addition to the diagnostic tools of the orthopedic surgeon.

  16. Selective inhibition by a synthetic hirudin peptide of fibrin-dependent thrombosis in baboons

    SciTech Connect

    Cadroy, Y.; Hanson, S.R.; Harker, L.A. ); Maraganore, J.M. )

    1991-02-15

    To determine the importance of the thrombin substrate recognition exosite for fibrinogen binding in the formation of both arterial and venous thrombi the authors evaluated the antithrombotic effects of the tyrosine-sulfated dodecapeptide from residues 53-64 of hirudin (H peptide) in a nonhuman primate model. This peptide was studied because it inhibits thrombin cleavages of fibrinogen by simple competition without blocking enzyme catalytic-site function. When an exteriorized arteriovenous access shunt model was used in baboons (Papio anubis), thrombus formation was induced by placing a thrombogenic device made of (i) a segment of tubing coated covalently with type I collagen, which generated platelet-rich thrombi under arterial flow conditions, and (ii) two subsequent annular regions of flow expansion that produced fibrin-rich thrombi typically associated with venous valves and veins. Thrombus formation was quantified by measurements of {sup 111}In-labeled platelet and {sup 125}I-labeled fibrinogen deposition in both arterial-flow and venous-flow portions of the device. These finding suggest that, by competitive inhibition of fibrinogen binding to thrombin, fibrin-rich venous-type thrombus formation may be selectively prevented. This strategy may be therapeutically attractive for preserving normal platelet function when conventional anticoagulant therapy is contraindicated.

  17. Development of a 124I-labeled version of the anti-PSMA monoclonal antibody capromab for immunoPET staging of prostate cancer: Aspects of labeling chemistry and biodistribution.

    PubMed

    Tolmachev, Vladimir; Malmberg, Jennie; Estrada, Sergio; Eriksson, Olof; Orlova, Anna

    2014-06-01

    Correct staging of prostate cancer is an unmet clinical need. Radionuclide targeting of prostate-specific membrane antigen (PSMA) with 111In-labeled capromab pendetide (ProstaScint) is a clinical option for prostate cancer staging. We propose the use of 124I-labeled capromab to decrease the retention of radioactivity in healthy organs (due to the non-residualizing properties of the radiolabel). The use of 124I as a label should increase imaging sensitivity due to the advantages of PET as an imaging modality. Capromab targets the intracellular domain of PSMA; accumulation of radioactivity in the tumor should not depend on internalization of the antigen/antibody complex. Capromab was iodinated, and its targeting properties were compared with indium labeled counterpart in LNCaP xenografts in dual isotope mode. PSMA-negative xenografts (PC3) were used as a negative control. Radioiodinated capromab bound to PSMA specifically. Biodistribution of 125I/111In-capromab showed a more rapid clearance of iodine radioactivity from liver, spleen, kidneys, bones, colon tissue, as well as tumors. Maximum tumor uptake (13±8% ID/g for iodine and 29±9% ID/g for indium) and tumor-to-non-tumor ratios for both agents were measured 5 days post-injection (pi). High tumor accumulation and low uptake of radioactivity in normal organs were confirmed using microPET/CT 5 days pi of 124I-capromab.

  18. Value of blood-pool subtraction in cardiac indium-111-labeled platelet imaging

    SciTech Connect

    Machac, J.; Vallabhajosula, S.; Goldman, M.E.; Goldsmith, S.J.; Palestro, C.; Strashun, A.; Vaquer, R.; Phillips, R.A.; Fuster, V. )

    1989-09-01

    Blood-pool subtraction has been proposed to enhance {sup 111}In-labeled platelet imaging of intracardiac thrombi. We tested the accuracy of labeled platelet imaging, with and without blood-pool subtraction, in ten subjects with cardiac thrombi of varying age, eight with endocarditis being treated with antimicrobial therapy and ten normal controls. Imaging was performed early after labeled platelet injection (24 hr or less) and late (48 hr or more). Blood-pool subtraction was carried out. All images were graded subjectively by four experienced, blinded readers. Detection accuracy was measured by the sensitivity at three fixed levels of specificity estimated from receiver operator characteristic curve analysis and tested by three-way analysis of variance. Detection accuracy was generally improved on delayed images. Blood-pool subtraction did not improve accuracy. Although blood-pool subtraction increased detection sensitivity, this was offset by decreased specificity. For this population studied, blood-pool subtraction did not improve subjective detection of abnormal platelet deposition by 111In platelet imaging.

  19. The use of indium-111 oxine platelet scintigraphy and survival studies in pediatric patients with thrombocytopenia

    SciTech Connect

    Castle, V.P.; Shulkin, B.L.; Coates, G.; Andrew, M. )

    1989-11-01

    We have utilized {sup 111}In-labeled heterologous platelets to investigate the mechanism of thrombocytopenia in ten children. From the scintigraphic findings, platelet survival times, and clinical information, thrombocytopenia was ascribed to decreased production or to increased destruction. Two patients were found to have bone marrow production defects. Two patients with hemangiomas were studied. In one, the hemangioma was shown not to be the cause of thrombocytopenia. In the second, the hemangioma was proven the source of platelet destruction, but was much more extensive than clinically evident. In both, surgical manipulation of the hemangioma was avoided. Six additional patients had thrombocytopenia due to accelerated destruction. In four, the spleen was shown responsible. In two, however, the spleen was shown not to be responsible for the low platelet counts, and splenectomy was avoided. Thus, {sup 111}In-platelet scintigraphy and survival studies are valuable in the classification and management of childhood thrombocytopenia. We believe that this study should be performed, when possible, in any child with thrombocytopenia where the mechanism is unclear or the therapeutic intervention involves splenectomy or resection of a hemangioma.

  20. Graft revascularization is essential for non-invasive monitoring of transplanted islets with radiolabeled exendin

    PubMed Central

    Eter, Wael A.; Bos, Desirée; Frielink, Cathelijne; Boerman, Otto C.; Brom, Maarten; Gotthardt, Martin

    2015-01-01

    Islet transplantation is a novel promising strategy to cure type 1 diabetes. However, the long-term outcome is still poor, because both function and survival of the transplant decline over-time. Non-invasive imaging methods have the potential to enable monitoring of islet survival after transplantation and the effects of immunosuppressive drugs on transplantation outcome. 111In-labeled exendin-3 is a promising tracer to visualize native and transplanted islets by SPECT (Single Photon Emission Computed Tomography). In the present study, we hypothesized that islet microvasculature plays an important role determining the uptake of exendin-3 in islets when monitoring transplant survival. We observed 111In-exendin-3 accumulation in the transplant as early as three days after transplantation and an increase in the uptake up to three weeks post-transplantation. Islet-revascularization correlated with the increase in 111In-exendin-3 uptake, whereas fully re-established islet vasculature coincided with a stabilized uptake of the radiotracer in the transplant. Here, we demonstrate the importance of islet vasculature for in vivo delivery of radiotracers to transplanted islets and we demonstrate that optimal and stable uptake of exendin four weeks after transplantation opens the possibility for long-term monitoring of islet survival by SPECT imaging. PMID:26490110

  1. Graft revascularization is essential for non-invasive monitoring of transplanted islets with radiolabeled exendin.

    PubMed

    Eter, Wael A; Bos, Desirée; Frielink, Cathelijne; Boerman, Otto C; Brom, Maarten; Gotthardt, Martin

    2015-10-22

    Islet transplantation is a novel promising strategy to cure type 1 diabetes. However, the long-term outcome is still poor, because both function and survival of the transplant decline over-time. Non-invasive imaging methods have the potential to enable monitoring of islet survival after transplantation and the effects of immunosuppressive drugs on transplantation outcome. (111)In-labeled exendin-3 is a promising tracer to visualize native and transplanted islets by SPECT (Single Photon Emission Computed Tomography). In the present study, we hypothesized that islet microvasculature plays an important role determining the uptake of exendin-3 in islets when monitoring transplant survival. We observed (111)In-exendin-3 accumulation in the transplant as early as three days after transplantation and an increase in the uptake up to three weeks post-transplantation. Islet-revascularization correlated with the increase in (111)In-exendin-3 uptake, whereas fully re-established islet vasculature coincided with a stabilized uptake of the radiotracer in the transplant. Here, we demonstrate the importance of islet vasculature for in vivo delivery of radiotracers to transplanted islets and we demonstrate that optimal and stable uptake of exendin four weeks after transplantation opens the possibility for long-term monitoring of islet survival by SPECT imaging.

  2. Imaging Lung Clearance of Radiolabeled Tumor Cells to Study Mice with Normal, Activated or Depleted Natural Killer (NK) Cells

    SciTech Connect

    Kulkarni, P.V.; Bennett, M.; Constantinescu, A.; Arora, V.; Viguet, M.; Antich, P.; Parkey, R.W.; Mathews, D.; Mason, R.P.; Oz, O.K.

    2003-08-26

    Lung clearance of 51CR and 125I iododeoxyuridine (IUDR) labeled cancer cells assess NK cell activity. It is desirable to develop noninvasive imaging technique to assess NK activity in mice. We labeled target YAC-1 tumor cells with 125I, 111In, 99mTc, or 67Ga and injected I.V. into three groups of BALB/c mice. Animals were treated with medium (group I), 300mg/kg cyclophosmamide (CY) to kill NK cell (group II), or anti-LY49C/1) (ab')2 mAb to augment NK function (group III). Lungs were removed 15 min or 2 h later for tissue counting. Control and treated mice were imaged every 5 min with a scintillating camera for 1 h after 15 min of infusion of the 111In labeled cells. Lung clearance increased after 15 min (lodging: 60-80%) and (2 h retention: 3-7%). Similar results were obtained with all the isotopes studied. Images distinguished the control and treated mice for lung activity. Cells labeled with 111In, 99mTc or 67Ga are cleared similar to those labeled with 51Cr or 125I. NK cell destruction of tumor cells may be assessed by noninvasive imaging method either by SPECT (99mTc, 111In, 67Ga) or by PET (68Ga)

  3. Extraction, radiolabeling, and in vivo catabolism of autologous-origin equine fibrinogen and platelets in the healthy and exercise-stressed horse

    SciTech Connect

    Coyne, C.P.

    1986-01-01

    Three separate techniques were evaluated for the extraction of autologous-origin fibrinogen from whole equine plasma. Rapid extraction of equine fibrinogen with ammonium sulfate-sodium phosphate buffer, in combination with saturated glycine buffer, provided the most practical means of obtaining a protein extract with the highest degree of biological activity and sufficiently high iodine-125 (/sup 125/I) radiolabeling efficiencies using monochloroiodine reagent (ICI). A technique was developed for the in vitro radiolabeling of equine platelets suspended in plasma. This entailed the use of the isotope, indium-111 (/sup 111/In), together with the lipophilic ligand, 2-(mercaptopyridine-N-oxide). This labeling technique achieved labeling efficiencies between 75% and 96%, and in vitro aggregability of /sup 111/In-merc radiolabeled platelets was comparable to that of unlabeled cell isolates. In the final phase of the investigation, autologous-origin /sup 125/I-labeled fibrinogen and /sup 111/In-labeled platelets were applied in a series of equine exercise physiology studies. Elimination of these two radiobiologicals was evaluated in the resting and exercise-stressed horse. Results from these investigations revealed no long-term influence of exercise conditioning on the in vivo kinetics of radiolabeled fibrinogen or platelets.

  4. Metabolic imaging with gallium-68- and indium-111-labeled low-density lipoprotein

    SciTech Connect

    Moerlein, S.M.; Daugherty, A.; Sobel, B.E.; Welch, M.J. )

    1991-02-01

    Low-density lipoprotein (LDL) labeled with either gallium-68 ({sup 68}Ga) or indium-111 ({sup 111}In) was evaluated as a potential PET or SPECT radiopharmaceutical for determination of hepatic lipoprotein metabolism in rabbits. Gallium-68 or {sup 111}In was linked to LDL via diethylenetriaminepentaacetic acid (DTPA) with a 25-70% radiochemical yield. Studies in vivo that compared {sup 68}Ga- or {sup 111}In-DTPA-LDL with dilactitol-({sup 125}I)-tyramine LDL and 131I-LDL showed that both {sup 68}Ga- and {sup 111}In-labeled LDL behaved as residualizing radiotracers. Localization of radioactivity within the liver of normal rabbits was visualized clearly with ({sup 68}Ga)DTPA-LDL by PET and with ({sup 111}In)DTPA-LDL by gamma scintigraphy. Significant differences were observed in hepatic uptake of normal compared with hypercholesterolemic rabbits in which low-capacity LDL receptor-mediated catabolism was saturated. Gallium-68 and {sup 111}In-DTPA-LDL are attractive radiopharmaceuticals for noninvasive delineation of tissue LDL metabolism under normal and pathophysiologic conditions.

  5. Cationorm shows good tolerability on human HCE-2 corneal epithelial cell cultures.

    PubMed

    Kinnunen, Kati; Kauppinen, Anu; Piippo, Niina; Koistinen, Arto; Toropainen, Elisa; Kaarniranta, Kai

    2014-03-01

    Preservatives have been for a long time known to cause detrimental effects on ocular surface. Cationorm, a preservative-free compound with electrostatic properties is a novel way to solve the problems encountered with traditional benzalkonium chloride (BAK)-containing eye drops. The aim of this study was to evaluate tolerability of the preservative-free cationic emulsion Cationorm in vitro on corneal epithelial cells. The human corneal epithelial cell (HCE-2) culture line was used to study cellular morphology, cytotoxicity and inflammatory responses after Cationorm diluted 1/10 exposure for 5, 15 and 30 min. Exposures to Systane diluted 1/10 with polyquaternium-1/polidronium chloride 0.001% as preservative, BAK 0.001% or C16 (0.0002%) and normal cell culture medium served as positive and negative references. Cell viability was determined by measuring the release of lactate dehydrogenase (LDH) and mitochondrial dehydrogenase activity was evaluated using 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The possible induction of apoptosis was analyzed by measuring the activity of caspase-3, and Cell Counting Kit-8 (CCK-8) was used to evaluate the number of viable cells after the exposure to test compounds. Furthermore, the tendency of the test compounds to produce inflammatory reaction was determined by analyzing the production of proinflammatory cytokines IL-6 and IL-8, and DNA binding of the p65 subunit of transcription factor NF-κB was measured from cell lysates. HCE-2 cells showed no morphological changes after the exposure to Cationorm, but in cells exposed to BAK, clear cytoplasm vacuolization and loose cell-cell contacts were observed in transmission (TEM) or scanning (SEM) electron microscopic analyses. Cell viability, as measured with the release of LDH, indicated a time dependent increase in LDH expression after exposure to all test compounds but especially with BAK. Moreover, Cationorm and BAK time-dependently decreased the

  6. Synthesis and biological evaluation of cholecystokinin analogs in which the Asp-Phe-NH2 moiety has been replaced by a 3-amino-7-phenylheptanoic acid or a 3-amino-6-(phenyloxy)hexanoic acid.

    PubMed

    Amblard, M; Rodriguez, M; Lignon, M F; Galas, M C; Bernad, N; Artis-Noël, A M; Hauad, L; Laur, J; Califano, J C; Aumelas, A

    1993-10-01

    Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester (JMV180), an analog of the C-terminal octapeptide of cholecystokinin (CCK-8), shows interesting biological activities behaving as an agonist at the high-affinity CCK binding sites and as an antagonist at the low-affinity CCK binding sites in rat pancreatic acini. Although we did not observe any major hydrolysis of the ester bond of Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester in our in vitro studies, we were aware of a possible and rapid cleavage of this ester bond during in vivo studies. To improve the stability of Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester, we decided to synthesize analogs in which the ester bond would be replaced by a carba (CH2-CH2) linkage. We synthesized the 3-amino-7-phenylheptanoic acid (beta-homo-Aph) with the R configuration in order to mimic the Asp-2-phenylethyl ester moiety and the 3-amino-6-(phenyloxy)hexanoic acid (H-beta-homo-App-OH), an analog of H-beta-homo-Aph-OH in which a methylene group has been replaced by an oxygen. (R)-beta-Homo-Aph and (R)-H-beta-homo-App-OH were introduced in the CCK-8 sequence to produce Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-(R)-beta-homo-Aph-OH and Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-(R)-beta-homo-App-OH. Both compounds were able to recognize the CCK receptor on rat pancreatic acini (IC50 = 12 +/- 8 nM and 13 +/- 5 nM, respectively), on brain membranes (IC50 = 32 +/- 2 nM and 57 +/- 5 nM, respectively), and on Jurkat T cells (IC50 = 75 +/- 15 nM and 65 +/- 21 nM, respectively). Like Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester, both compounds produced maximal stimulation of amylase secretion (EC50 = 6 +/- 2 nM and 4 +/- 2 nM, respectively) with no decrease of the secretion at high concentration indicating that these compounds probably act as agonists at the high-affinity peripheral CCK-receptor and as antagonists at the low-affinity CCK-receptor. Replacing the tryptophan by a D-tryptophan in such analogs produced full CCK

  7. [Reversal of adriamycin resistance by digoxin in human breast cancer cell line MCF-7/adriamycin and its mechanism].

    PubMed

    Li, Bai-He; Yuan, Lei; Shi, Ran-Ran; Wang, Jian-Guo

    2015-12-25

    The aim of this study was to investigate the effects of digoxin on the chemoresistance of human breast cancer cell line MCF-7/adriamycin (ADR) and its underlying mechanism. MCF-7 and MCF-7/ADR cells were designated as control and ADR groups, respectively. MCF-7/ADR cells in ADR + digoxin group received 48 h of digoxin (10 nmol/L) treatment; MCF-7/ADR cells transfected with pLKO.1-shHIF-1α and pLKO.1-shcontrol plasmids were named shHIF-1α and shcontrol groups, respectively. CCK-8 assay was employed to detect the cytotoxic effect of ADR on MCF-7/ADR cells, and IC50 value and resistance index were calculated according to CCK-8. RT-PCR was used to measure the mRNA levels of hypoxia inducible factor-1α (HIF-1α) and multidrug resistance-1 (MDR1). Western blot was used to analyze the protein levels of HIF-1α and MDR1. Flow cytometry was used to determine the apoptosis. The result showed that the resistance index of MCF-7/ADR cells was 115.6, and it was reduced to 47.2 under the action of digoxin (P < 0.05). In comparison with control group, ADR groups showed increased protein and mRNA levels of HIF-1α and MDR1 (P < 0.05). Digoxin reduced the protein levels of HIF-1α and MDR1, as well as the mRNA level of MDR1, but did not affect the mRNA level of HIF-1α. After HIF-1α gene was silenced, the protein levels of HIF-1α and MDR1 were down-regulated (P < 0.05), and the pro-apoptotic effect of ADR on MCF-7/ADR cells was enhanced. Although it was also observed that digoxin promoted cell apoptosis in both shcontrol and shHIF-1α groups, the difference between the two groups was not significant. In conclusion, the results suggest that digoxin may partially reverse the ADR resistance in human breast cancer cell line MCF-7/ADR by means of down-regulating the expression levels of HIF-1α and MDR1 and promoting apoptosis via HIF-1α-independent pathway.

  8. [Experimental study on tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid in vitro].

    PubMed

    Yang, Zelong; Chen, Zhu; Liu, Kang; Bai, Yiguang; Jiang, Ting; Feng, Daxiong; Feng, Gang

    2013-10-01

    To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning. The three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA (8 : 1, W : W), which was dissolved in mixed solvent of 3-trifluoroethanol and water (1 : 1, V : V). The morphology were observed by light microscope and scanning electron microscope (SEM). And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 (CCK-8). The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix (ECM) secretion were observed by histological analysis. The optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% +/- 25.0%. The contact angle was (35.6 +/- 3.4) degrees, and the water absorption was 1 120% +/- 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% +/- 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% +/- 11.26% at 12 hours, and the cell survival rate was 126.03% +/- 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. The

  9. CD147 promotes the proliferation, invasiveness, migration and angiogenesis of human lung carcinoma cells

    PubMed Central

    Yang, Shaoxing; Qi, Fei; Tang, Chuanhao; Wang, Hong; Qin, Haifeng; Li, Xiaoyan; Li, Jianjie; Wang, Weixia; Zhao, Changyun; Gao, Hongjun

    2017-01-01

    Cluster of differentiation (CD) 147 is a transmembrane glycoprotein that is highly expressed at the tumor cell surface, which stimulates fibroblasts to produce a large number of matrix metalloproteinases and promotes tumor invasion and metastasis and tumor-induced angiogenesis. The present study investigated the functions and the role of CD147 in the human lung carcinoma A549 cell line. The present study constructed expression and interference [small interfering (si) RNA] lentiviral vectors of CD147, which established stable overexpression and low expression of CD147 in the A549 cell line, named A549-CD147 and A549-siCD147, respectively. The differences in biological features between various levels of CD147 expression in A549 cells was investigated by cell counting kit-8 (CCK-8), Transwell, scratch and lumen formation assays. The results of the CCK-8 assay revealed that A549-CD147 cell proliferation was significantly increased and A549-siCD147 cell proliferation was decreased compared with the control groups. The A549-CD147 cells had the largest number of cells penetrating the Matrigel in the Transwell assay, which indicates that upregulation of CD147 expression increases the infiltration capacity of cells. The scratch assay revealed that A549-CD147 cells have the highest capacity for migration, while A549-siCD147 cells have the lowest. Quantitative polymerase chain reaction and western blot analysis demonstrated that vascular endothelial growth factor (VEGF) expression was proportional to the expression level of CD147 at the mRNA and protein level. The lumen formation assay revealed that the number of vessel lumens that human umbilical vein endothelial cells formed in the A549-CD147 cell supernatant was increased compared with the A549-siCD147 cells. Collectively, the present results suggest that CD147 is important in the promotion of lung carcinoma cell proliferation, invasion and metastasis and the upregulation of VEGF, which stimulates the angiogenesis of lung

  10. In vitro anticancer properties of selected Eucalyptus species.

    PubMed

    Bhuyan, Deep Jyoti; Sakoff, Jennette; Bond, Danielle R; Predebon, Melanie; Vuong, Quan V; Chalmers, Anita C; van Altena, Ian A; Bowyer, Michael C; Scarlett, Christopher J

    2017-08-01

    In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI50 values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC50 values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.

  11. [Suppression of Aurora-A by RNA interference inhibits laryngeal cancer Hep-2 cell growth].

    PubMed

    Zhang, Hao; Chen, Xue-hua; Cai, Chang-ping; Wang, Shi-li; Liu, Bing-ya; Zhou, Liang

    2012-01-01

    To investigate the effects of knockdown of Aurora-A by RNA interference on laryngeal cancer Hep-2 cell growth in vitro and in vivo. A plasmid containing siRNA against Aurora-A was constructed and transfected into human laryngeal cancer cell line Hep-2. Measurements included the CCK-8 assay for viability and proliferation, Transwell assay for invasion, colony formation assay for cell anchorage-independent growth. Western blot and immunohistochemistry assay for protein expression. Tumorigenicity was observed in vivo. In Hep-2 cells transfected by Aurora-A siRNA (designated as siRNA-3), protein expression of Aurora-A was suppressed by 52%. In CCK-8 assay, absorbance value of siRNA-3 cells (3.268 ± 0.106, (x(-) ± s)) was lower than that of Hep-2 cells (3.722 ± 0.152, F = 17.634, P < 0.001). In Transwell assay, the average invasive cells per field in siRNA-3 cells (110.0 ± 18.0) was less than that in Hep-2 cells (236.0 ± 26.0, F = 26.462, P < 0.01). In colony formation assay, the average colony number of siRNA-3 cells (31.0 ± 6.6) was lower than that of Hep-2 cells (104.0 ± 14.0). The average tumor size in siRNA-3 group was (127.77 ± 174.83) mm(3), which was less than Hep-2 cell group (837.26 ± 101.80) mm(3), (F = 28.187, P < 0.001). Silencing of Aurora-A decreased the expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2), key regulators in cell adhesion and invasion. The knockdown of Aurora-A inhibits the growth and invasiveness of Hep-2 cells in vitro and in vivo, which may be a promising therapeutic strategy for LSCC.

  12. RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109.

    PubMed

    Zhang, Heping; Li, Jiancheng; Cheng, Wenfang; Liu, D I; Chen, Cheng; Wang, Xiaoying; Lu, Xujing; Zhou, Xifa

    2015-09-01

    The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity

  13. Antibacterial properties of nano-silver coated PEEK prepared through magnetron sputtering.

    PubMed

    Liu, Xiuju; Gan, Kang; Liu, Hong; Song, Xiaoqing; Chen, Tianjie; Liu, Chenchen

    2017-09-01

    We aimed to investigate the cytotoxicity and antibacterial properties of nano-silver-coated polyetheretherketone (PEEK) produced through magnetron sputtering and provide a theoretical basis for its use in clinical applications. The surfaces of PEEKs were coated with nano-silver at varying thicknesses (3, 6, 9, and 12nm) through magnetron sputtering technology. The resulting coated PEEK samples were classified into the following groups according to the thickness of the nano-silver coating: PEEK-3 (3nm), PEEK-6 (6nm), PEEK-9 (9nm), PEEK-12 (12nm), and PEEK control group. The surface microstructure and composition of each sample were observed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy dispersive spectrum (EDS) analysis. The water contact angle of each sample was then measured by contact angle meters. A cell counting kit (CCK-8) was used to analyze the cytotoxicity of the mouse fibroblast cells (L929) in the coated groups (n=5) and group test samples (n=6), negative control (polyethylene, PE) (n=6), and positive control group (phenol) (n=6). The antibacterial properties of the samples were tested by co-culturing Streptococcus mutans and Straphylococcus aureus. The bacteria that adhered to the surface of samples were observed by SEM. The antibacterial adhesion ability of each sample was then evaluated. SEM and AFM analysis results showed that the surfaces of control group samples were smooth but compact. Homogeneous silver nano-particles (AgNPs) and nano-silver coating were uniformly distributed on the surface of the coated group samples. Compared with the control samples, the nano-silver coated samples had a significant increase in surface roughness (P<0.05) as the thickness of their nano-silver coating increased. EDS analysis showed that not only C and O but also Ag were present on the surface of the coated samples. Moreover, the water contact angle of modified samples significantly increased after nano-silver coating modification

  14. Preliminary screening of differentially expressed genes involved in methyl-CpG-binding protein 2 gene-mediated proliferation in human osteosarcoma cells.

    PubMed

    Meng, Gang; Li, Yi; Lv, YangFan; Dai, Huanzi; Zhang, Xi; Guo, Qiao-Nan

    2015-04-01

    Methyl-CpG-binding protein 2 (MeCP2) is essential in human brain development and has been linked to several cancer types and neuro-developmental disorders. This study aims to screen the MeCP2 related differentially expressed genes and discover the therapeutic targets for osteosarcoma. CCK8 assay was used to detect the proliferation and SaOS2 and U2OS cells. Apoptosis of cells was detected by flow cytometry analysis that monitored Annexin V-APC/7-DD binding and 7-ADD uptake simultaneously. Denaturing formaldehyde agarose gel electrophoresis was employed to examine the quality of total RNA 18S and 28S units. Gene chip technique was utilized to discover the differentially expressed genes correlated with MeCP2 gene. Differential gene screening criteria were used to screen the changed genes. The gene up-regulation or down-regulation more than 1.5 times was regarded as significant differential expression genes. The CCK8 results indicated that the cell proliferation of MeCP2 silencing cells (LV-MeCP2-RNAi) was significantly decreased compared to non-silenced cells (LV-MeCP2-RNAi-CN) (P < 0.05). MeCP2 silencing could also induce significant apoptosis compared to non-silenced cells (P < 0.05); 107 expression changed genes were screened from a total of 49,395 transcripts. Among the total 107 transcripts, 34 transcripts were up-regulated and 73 transcripts were down-regulated. There were five significant differentially expressed genes, including IGFBP4, HOXC8, LMO4, MDK, and CTGF, which correlated with the MeCP2 gene. The methylation frequency of CpG in IGFBP4 gene could achieve 55%. In conclusion, the differentially expressed IGFBP4, HOXC8, LMO4, MDK, and CTGF genes may be involved in MeCP2 gene-mediated proliferation and apoptosis in osteosarcoma cells.

  15. TFP5 prevents 1-methyl-4-phenyl pyridine ion-induced neurotoxicity in mouse cortical neurons

    PubMed Central

    Zhang, Qi-Shan; Liao, Yuan-Gao; Ji, Zhong; Gu, Yong; Jiang, Hai-Shan; Xie, Zuo-Shan; Pan, Su-Yue; Hu, Ya-Fang

    2016-01-01

    The present study aimed to investigate the protective effect of a modified p5 peptide, TFP5, on 1-methyl-4-phenyl pyridine ion (MPP+)-induced neurotoxicity in cortical neurons and explore the therapeutic effect of TFP5 on Parkinson's disease (PD). MPP+ was applied to a primary culture of mouse cortical neurons to establish the cell model of PD. Neurons were divided into four groups: Control, model (MPP+), scrambled peptide (Scb) (Scb + MPP+) and TFP5 (TFP5 + MPP+) groups. Pretreatment with Scb or TFP5 was applied to the latter two groups, respectively, for 3 h, while phosphate-buffered saline was applied to the control and model groups. MPP+ was then applied to all groups, with the exception of the control group, and neurons were cultured for an additional 24 h. Neuron viability was evaluated using a Cell Counting kit-8 (CCK8) assay. To explore the mechanism underlying the protective effects of TFP5, the expression levels of p35, p25 and phosphorylated myocyte enhancer factor 2 (p-MEF2D) were determined by western blotting. Fluorescence microscopy showed that TFP5 was able to pass through cell membranes and distribute around the nucleus. CCK8 assay showed that neuronal apoptosis was dependent on MPP+ concentration and exposure time. Cell viability decreased significantly in the model group compared with the control group (55±7 vs. 100±0%; P<0.01), and increased significantly in the TFP5 group compared with the model group (98±2 vs. 55±5%; P<0.01) and Scb group (98±2 vs. 54±4%; P<0.01). Scb exhibited no protective effect. Western blotting results showed that MPP+ induced p25 and p-MEF2D expression, TFP5 and Scb did not affect MPP+-induced p25 expression, but TFP5 reduced MPP+-induced p-MEF2D expression. In summary, TFP5 protects against MPP+-induced neurotoxicity in mouse cortical neurons, possibly through inhibiting the MPP+-induced formation and elevated kinase activity of a cyclin-dependent kinase 5/p25 complex. PMID:27698762

  16. CdSe/ZnS quantum dots induce photodynamic effects and cytotoxicity in pancreatic cancer cells

    PubMed Central

    He, Si-Jia; Cao, Jia; Li, Yong-Sheng; Yang, Jia-Chun; Zhou, Min; Qu, Chun-Ying; Zhang, Yi; Shen, Feng; Chen, Ying; Li, Ming-Ming; Xu, Lei-Ming

    2016-01-01

    AIM: To investigate the photodynamic effect of CdSe/ZnS quantum dots (QDs) on pancreatic cancer cells and elucidate the probable mechanisms. METHODS: The pancreatic cancer cell line SW1990 was treated with different concentrations of CdSe/ZnS QDs (0, 0.5, 1.0, 1.5, 2.0, 2.5 μmol/L), with or without illumination. The viability of SW1990 cells was tested using the Cell Counting Kit-8 (CCK-8) assay. The ultrastructural changes of SW1990 cells were observed by transmission electron microscopy. Apoptosis was detected by nuclear staining and flow cytometry (FCM). Reactive oxygen species (ROS) were measured by dichlorofluorescein diacetate via fluorescence microscopy. Expression of Bax, Bcl-2 and caspase-3 was measured by real-time polymerase chain reaction (PCR) and protein immunoblotting 24 h after SW1990 cells were treated with CdSe/ZnS QDs and illuminated. RESULTS: The CCK-8 assay results showed that both CdSe/ZnS QDs with and without illumination suppressed SW1990 cell proliferation. Cell viability was significantly lower when illuminated or with a longer incubation time and a higher light dose. CdSe/ZnS QDs with illumination caused ultrastructural changes in SW1990 cells, such as organelle degeneration and chromatin condensation and aggregation at the periphery of the nucleus. Fluorescence microscopy and FCM showed that CdSe/ZnS QDs (1.5 μmol/L) with illumination increased SW1990 cell apoptosis (53.2%) and ROS generation compared with no illumination. Real-time PCR showed that expression of Bax and caspase-3 was upregulated and Bcl-2 was downregulated. Immunoblotting results were consistent with real-time PCR results. Inhibition of ROS and apoptosis both attenuated QD-photodynamic-therapy-induced cell death. CONCLUSION: CdSe/ZnS QDs can be used as a photosensitizer to inhibit SW1990 cell proliferation through ROS generation and apoptotic protein expression regulation. PMID:27275093

  17. [Influence of BMP-7 on chondrocyte secretion and expression of Col-II,AGG and Sox9 mRNA in porous tantalum-chondrocyte composites in vitro].

    PubMed

    Zhang, H; Li, L; Wang, Q; Gan, H Q; Wang, H; Bi, C; Li, Q J; Wang, Z Q

    2015-04-18

    To study the influence of bone morphogenetic protein-7 (BMP-7) on chondrocyte secretion and expression of type II collagen (Col-II), aggrecan (AGG) and SRY-related high mobility group-box gene 9 (Sox9) mRNA in porous tantalum-chondrocyte composites. The articular chondrocytes were isolated from 3-week-old New Zealand immature rabbits and identified. The 2nd generation of chondrocytes with 1×10(6)/mL inoculate concentration was seeded in porous tantalum and divided into 4 groups, and control group (tantalum/chondrocyte), 50 μg/L BMP-7 group (50 μg/L BMP-7/tantalum/chondrocyte), 100 μg/L BMP-7 group (100 μg/L BMP-7/tantalum/chondrocyte), and 200 μg/L BMP-7 group (200 μg/L BMP-7/tantalum/chondrocyte). The proliferation of chondrocytes was measured by CCK-8 assay. The chondrocyte growth and morphology were observed by scanning electron microscopy (SEM). The synthesis of glycosaminoglycan (GAG) in chondrocytes was tested by dimethyl methylene blue (DMMB) colorimetric quantification method. Col-II, AGG and Sox9 mRNA in chondrocytes were detected by real-time PCR. The chondrocytes were spindle-shaped in 24 hours of primary cell culture and most cells became polygonal shaped in 4 days. The chondrocytes were affirmed by alcian blue, safranin O and Col-II immunocytochemistry staining. The result of CCK-8 assay showed that the level of cell proliferation in 100 μg/L BMP-7 groups were higher than those in the other groups (P<0.05). The chondrocytes implanted into porous tantalum scaffolds with BMP-7 had better functions, by which cytoplasmic processes developed and extended to the surface and inner of porous tantalum by SEM observation. DMMB quantitative determination of GAG showed that GAG amount of chondrocytes in 100 μg/L BMP-7 groups was significantly higher than those in the other groups (P<0.05). The expressions of Col-II, AGG and Sox9 mRNA in chondrocytes were up-regulated in the experimental groups, compared with the control group and the best effect appeared

  18. Reversion of trichostatin A resistance via inhibition of the Wnt signaling pathway in human pancreatic cancer cells.

    PubMed

    Wang, Benquan; Zou, Qian; Sun, Meng; Chen, Jingfeng; Wang, Tianyang; Bai, Yongheng; Chen, Zongjing; Chen, Bicheng; Zhou, Mengtao

    2014-11-01

    Drug resistance is a major impediment to successful chemotherapy in pancreatic cancer (PC) patients. We investigated the effect of Wnt/β-catenin signaling inhibition by wnt-c59 on chemoresistance in a trichostatin A-resistant Panc-1 cell line (Panc-1/TSA). Panc-1/TSA cells were treated with the Wnt/β‑catenin signaling inhibitor wnt-c59 (10 µmol · l-1) and/or trichostatin A (TSA; 10 µmol · l-1) for 24 h. CCK-8 assay was utilized to analyze the interactive effect of TSA and wnt-c59 on induction of apoptosis of the Panc-1/TSA cells. Cell apoptosis was measured by flow cytometry. Real-time PCR and western blotting were used to assess Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT) and multidrug resistance (MDR). Real-time cell analysis (RTCA) was used to detect the cell migration ability. After wnt-c59 treatment for 24 h, relative genes and transcriptional targets of Wnt/β-catenin signaling were downregulated (P<0.05). CCK-8 assay indicated that the combination of TSA and wnt-c59 had a synergistic effect on induction of Panc-1/TSA cell apoptosis. As detected by FACS, cell apoptosis rates increased significantly (P<0.05). The results of RTCA showed that the cell indices of the control group, wnt-c59 group, TSA group and TSA+wnt-c59 combination group were 1.2842±0.0257, 1.2155±0.0282, 1.2533±0.0194 and 0.8541±0.0250, respectively. In accordance, MMP-9 protein in the wnt-c59 treatment groups was decreased compared to the non-wnt-c59 treatment groups. Meanwhile, E-cadherin protein was upregulated and vimentin protein was downregulated, both of which are characteristic markers of EMT. Chemoresistant gene MDR1 and P-glycoprotein (P-gp) in the wnt-c59 treatment groups had a reduced expression compared to the non-wnt-c59 treatment groups. This study revealed that TSA sensitivity, migration ability, and the EMT phenotype in Panc-1/TSA cells were reversed following Wnt/β-catenin signaling inhibition.

  19. Gold nanorods as a theranostic platform for in vitro and in vivo imaging and photothermal therapy of inflammatory macrophages

    NASA Astrophysics Data System (ADS)

    Qin, Jinbao; Peng, Zhiyou; Li, Bo; Ye, Kaichuang; Zhang, Yuxin; Yuan, Fukang; Yang, Xinrui; Huang, Lijia; Hu, Junqing; Lu, Xinwu

    2015-08-01

    Inflammatory macrophages play pivotal roles in the development of atherosclerosis. Theranostics, a promising approach for local imaging and photothermal therapy of inflammatory macrophages, has drawn increasing attention in biomedical research. In this study, gold nanorods (Au NRs) were synthesized, and their in vitro photothermal effects on the macrophage cell line (Ana-1 cells) under 808 nm near infrared reflection (NIR) were investigated by the CCK8 assay, calcein AM/PI staining, flow cytometry, transmission electron microscopy (TEM), silver staining and in vitro micro-computed tomography (CT) imaging. These Au NRs were then applied to an apolipoprotein E knockout (Apo E) mouse model to evaluate their effects on in vivo CT imaging and their effectiveness as for the subsequent photothermal therapy of macrophages in femoral artery restenosis under 808 nm laser irradiation. In vitro photothermal ablation treatment using Au NRs exhibited a significant cell-killing efficacy of macrophages, even at relatively low concentrations of Au NRs and low NIR powers. In addition, the in vivo results demonstrated that the Au NRs are effective for in vivo imaging and photothermal therapy of inflammatory macrophages in femoral artery restenosis. This study shows that Au nanorods are a promising theranostic platform for the diagnosis and photothermal therapy of inflammation-associated diseases.Inflammatory macrophages play pivotal roles in the development of atherosclerosis. Theranostics, a promising approach for local imaging and photothermal therapy of inflammatory macrophages, has drawn increasing attention in biomedical research. In this study, gold nanorods (Au NRs) were synthesized, and their in vitro photothermal effects on the macrophage cell line (Ana-1 cells) under 808 nm near infrared reflection (NIR) were investigated by the CCK8 assay, calcein AM/PI staining, flow cytometry, transmission electron microscopy (TEM), silver staining and in vitro micro-computed tomography

  20. In Vitro Effects of Preservative-free and Preserved Prostaglandin Analogs on Primary Cultured Human Conjunctival Fibroblast Cells

    PubMed Central

    Kim, Eun Joo; Kim, Yeoun-Hee; Kang, Sun-Hee; Lee, Kyoo Won

    2013-01-01

    Purpose Long-term use of topical medication is needed for glaucoma treatment. One of the most commonly prescribed classes of hypotensive agents are prostaglandin analogs (PGs) used as both first-line monotherapy; as well as in combination therapy with other hypotensive agents. Several side effects of eye drops can be caused by preservatives. The purpose of this study was to evaluate the effects of PGs with varying concentrations of benzalkonium chloride (BAC), alternative preservatives, or no preservatives on human conjunctival fibroblast cells. Methods Primary human conjunctival fibroblast cells were used in these experiments. Cells were exposed to the following drugs: BAC at different concentrations, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), tafluprost 0.0015% with/without 0.001% BAC and travoprost 0.004% (with 0.001% Polyquad) for 15 and 30 minutes. Cell cytotoxicity was evaluated by phase-contrast microscopy to monitor morphological changes of cells, Counting Kit-8 (CCK-8) assay to cell viability, and fluorescent activated cell sorting (FACS) analysis to measure apoptosis. Results BAC caused cell shrinkage and detachment from the plate in a dose-dependent manner. Morphological changes were observed in cells treated with bimatoprost 0.01% and latanoprost 0.005%. However, mild cell shrinkage was noted in cells treated with tafluprost 0.0015%, while a non-toxic effect was noted with travoprost 0.004% and preservative-free tafluprost 0.0015%. CCK-8 assay and FACS analysis showed all groups had a significantly decreased cell viability and higher apoptosis rate compared with the control group. However, travoprost 0.004% and preservative-free tafluprost 0.0015% showed lower cytotoxicity and apoptosis rate than other drugs. Conclusions This in vitro study revealed that BAC-induced cytotoxicity is dose-dependent, although it is important to emphasize that the clinical significance of toxicity differences observed among the different PGs

  1. [Influence of I2PP2A gene silencing by RNA interference on proliferation and apoptosis of human acute promyelocytic leukemia cell line NB4-R1].

    PubMed

    Liu, Yanfeng; He, Pengcheng; Liu, Feng; Cheng, Xiaoyan; Zhang, Mei

    2014-08-01

    To explore the effect of RNA interference of human I2PP2A gene on the proliferation and apoptosis of retinoic acid-resistant human acute promyelocytic leukemia (APL) cell line NB4-R1. Designed and constructed a RNA interference lentiviral vector I2PP2A-shRNA which targeted against I2PP2A gene, then transfected it into NB4-R1 via polybrene mediation. The I2PP2A expression levels before and after transfection were detected by qRT-PCR and Western blot, respectively. Meanwhile, the proliferation and apoptosis rates of each group were determined by CCK-8 and flow cytometry assay. The protein expressions of caspase-8 and PARP were detected by Western blot. Both qRT-PCR and Western blot data showed the I2PP2A expression level was significantly downregulated in the transfection group. The I2PP2A mRNA expression level decreased by (70.0 ± 9.6)% and (64.0 ± 6.2)% respectively, compared with blank control and negative control group, and the I2PP2A protein expression level showed a consistent trend. CCK-8 proliferation assay indicated the NB4-R1 cell proliferation rate in I2PP2A-shRNA transfection group significantly reduced compared to blank control group (P<0.05). Flow cytometry results showed that NB4-R1 apoptosis rate in I2PP2A-shRNA transfection group increased by (6.30 ± 0.67) times and (6.04 ± 0.56) times, respectively (P<0.01). After inhibition of I2PP2A, the total caspase-8 and total PARP expressions decreased by (44.0 ± 3.1)% and (57.0 ± 4.0)%, respectively; Meanwhile, the cleaved caspase-8 (p43) and cleaved PARP (p89) increased by (36.0 ± 2.5)% and (45.0 ± 4.8)%, respectively compared with blank control group (P<0.05). I2PP2A gene silenced by RNA interference could inhibit the proliferation and promote the apoptosis of NB4-R1, which may be regulated through caspase-8-induced exogenous apoptosis pathway.

  2. Immunomodulatory proteins FIP-gts and chloroquine induce caspase-independent cell death via autophagy for resensitizing cisplatin-resistant urothelial cancer cells.

    PubMed

    Hsin, I-Lun; Wang, Shao-Chuan; Li, Jian-Ri; Ciou, Tsai-Chun; Wu, Chih-Hsien; Wu, Hung-Ming; Ko, Jiunn-Liang

    2016-12-01

    Chloroquine, a lysosomal inhibitor, is used for malaria, rheumatoid arthritis, and lupus erythematosus therapy. In our previous study, FIP-gts, an immunomodulatory protein from Ganoderma tsugae, inhibited cell viability in lung cancer cells and urothelial cancer cells. Urothelial carcinoma is the most common type of bladder cancer. Cisplatin resistance is an important issue in urothelial carcinoma therapy. The aim of this study is to investigate the effect of combination treatment with FIP-gts and chloroquine on cytotoxicity to resensitize the cisplatin-resistant cells. FIP-gts and chloroquine cytotoxicity were determined by evaluating CCK-8 assay. Cell death pathways, ROS and cell cycle arrested were analysed through flow cytometry and Western blot. ShRNA targeting to autophagy-related genes were tested to evaluate their autophagic cell death for resistant urothelial cells. Using CCK-8 assay, chloroquine increased FIP-gts-induced cytotoxicity in parental and cisplatin-resistant urothelial cancer cell lines. On flow cytometry, chloroquine enhanced FIP-gts-mediated sub-G1 accumulation, annexin V positive signal and mitochondrial membrane potential loss. Caspase-3/PARP cascade and z-VAD-fmk were performed to prove that FIP-gts and chloroquine induced caspase-independent cell death. Using H2DCFDA staining and flow cytometry, FIP-gts and chloroquine did not induce ROS production. N-acetyl cysteine, a ROS scavenger, inhibited the cytotoxicity and LC3-II accumulation in FIP-gts and chloroquine-treated N/P cells. To elucidate the role of autophagy in caspase-independent cell death by FIP-gts and chloroquine, LC3 shRNA were used to inhibit autophagy in N/P cells. The capabilities of FIP-gts and chloroquine to induce cytotoxicity and sub-G1 phase accumulation were abolished in autophagy-defective cells. This is the first study to reveal the novel function of FIP-gts in triggering caspase-independent cell death in cisplatin-resistant urothelial cancer cells. Chloroquine

  3. Effects of Low-dose Triamcinolone Acetonide on Rat Retinal Progenitor Cells under Hypoxia Condition

    PubMed Central

    Xing, Yao; Cui, Li-Jun; Kang, Qian-Yan

    2016-01-01

    Background: Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition. Methods: RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48–72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3. Results: CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05). Conclusion: Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation. PMID:27364798

  4. [Parathyroid hormone inhibits the apoptosis of osteoblast MC-3T3E1 cells through a non-PLC-dependent protein kinase C pathway].

    PubMed

    Hu, Shao-Yu; Tong, Guo-Jun; Meng, Yue; Hao, Song; Li, Wei; Xu, Fu-Long; He, You-Hua; Chen, Jian-Ting; Yang, De-Hong

    2016-06-01

    To investigate the effect of the non-PLC-dependent protein kinase C (PKC) pathway of parathyroid hormone (PTH) on the apoptosis and proliferation of osteoblast MC-3T3E1 cells. MC-3T3E1 cells were seeded in 96-well plates at the density of 1.5×10(4) cells/mL and incubated for 3 day. The cells were then exposed to 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-28), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34)+1 µmol/L Go6983, 1 µmol/L Go6983, or deionized water (control) for 1, 24 or 48 h. After the treatments, cell counting kit-8 (CCK-8) and Caspase-Glo® 3/7 Assay (Caspase-3) were used to examine the proliferation and apoptosis of MC3T3-E1 cells. CCK-8 results showed that hPTH(1-34) increased the number of MC3T3-E1 cells compared with hPTH(1-34)+Go6983 at 1 h and 24 h, but this difference was not statistically different. At 48 h, treatment with hPTH(1-34), as compared with hPTH(1-28), significantly increased the number of MC3T3-E1 cells (P<0.05), and this effect was blocked by the PKC inhibitor Go6983 (P<0.05). hPTH(1-34) did not result in significant inhibition of MC3T3-E1 cell apoptosis at 1 h and 24 h as compared with hPTH(1-34)+Go6983, but significantly inhibited the cell apoptosis as compared with hPTH(1-28) (P<0.05); this inhibitory effect was blocked by Go6983 (P<0.05). s A relatively long time (for 48 h) of exposure to PTH can inhibit apoptosis and promote the proliferation of MC3T3-E1cells through a non-PLC-dependent PKC pathway.

  5. [Effects of component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on cell proliferation, apoptosis and skeleton in lung cancer A549 cells].

    PubMed

    Yan, Xiao-jing; Yang, Ye; Bi, Lei; Chen, Shan-shan; Zhu, Jing-jing; Chen, Wei-ping

    2014-11-01

    This study aims to optimize the most effective component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on lung cancer A549 using the orthogonal design method, and to investigate its effects of the component formula on cell proliferation, apoptosis and cytoskeleton in lung cancer A549 cells. The orthogonal design method was introduced to optimize the most effective component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on lung cancer A549 cells. CCK-8 assay and Real-time cell analysis were adapted to analyze the effect of component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma on A549 cells viability at different time and dose. Cell apoptosis was measured by Annexin V- FITC/PI double staining and flow cytometry. Cell skeleton protein F-actin was detected by high content screening (HCS). The optimizing component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma for total salvianolic acid, total saponins of panax ginseng and ginseng polysaccharide doses were 5, 10, 5 mg L(-1). CCK-8 assay and real-time cell analysis demonstrated that the component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma treatment could significantly decrease the A549 cell viability in both dose- and time-dependent manner compared with control group (P < 0.01). Moreover, the increase of cell apoptosis was detected by Annexin V-FITC/PI double staining and flow cytometry when cells treated with the component formula, which indicating that the component formula of Salviae Miltiorrhizae Radix et Rhizoma and Ginseng Radix et Rhizoma could induce A549 cell apoptosis in a time-dependent manner compared with control group (P < 0.01). Furthermore, compared with control group, a significant decrease in A549 cell skeleton area was found in the component formula-exposed cells in the dose-dependent manner (P < 0.01). In summary, the component formula

  6. 17β-Estradiol Inhibites Tumor Necrosis Factor-α Induced Apoptosis of Human Nucleus Pulposus Cells via the PI3K/Akt Pathway

    PubMed Central

    Wang, Tao; Yang, Si-Dong; Liu, Sen; Wang, Hui; Liu, Huan; Ding, Wen-Yuan

    2016-01-01

    Background Tumor necrosis factor-α (TNF-α) has been widely known to induce degeneration of nucleus pulposus cells (NPCs). 17β-estradiol (17β-E2) has been broadly proven for its function of suppressing cell apoptosis. The aim of this study is to explore whether 17β-E2 protects apoptosis of human NPCs induced by TNF-α via the PI3K/AKT pathway. Material/Methods NPCs were divided into four groups: control, TNF-α (100 ng/mL), TNF-α (100 ng/mL) with pretreated 17β-E2 (10 um/L), TNF-α (100 ng/mL) with pretreated 17β-E2 (10 um/L) and MK2206 (10 um/L, inhibitor of the PI3K/AKT pathway). Flow cytometry was used to measure the apoptotic incidence. Inverted phase-contrast microscopy was used to accomplish the morphological observation for apoptosis of treated cells. Additionally, Cell Counting Kit 8 (CCK-8) assay was used to detected cell proliferation. Western blot and quantitative real-time PCR (qRT-PCR) were applied to explore the expression of pro-caspase-3, caspase-3/p17, cleaved PARP, PARP, Akt, and phospho-Akt (p-Akt). Results First, inverted phase-contrast microscopy, CCK-8, and flow cytometry showed that TNF-α induced marked apoptosis, which was abolished by 17β-E2. Furthermore, Western blot and qRT-PCR showed that 17β-E2 protects TNF-α which can induced apoptosis by upregulating p-Akt, whereas Akt was essentially constant. Our data revealed that p-Akt expression peaked at 24 hours in a time-dependent manner (0–48 hours) after treating with TNF-α; and the p-Akt expression generally increased in a time-dependent manner (0–48 hours) after treating with TNF-α and 17β-E2. Conclusions 17β-E2 is shown to protect NPCs against TNF-α induced apoptosis by upregulating p-Akt in the PI3K/AKT pathway. 17β-E2 generally increases expression of p-Akt. PMID:27847386

  7. Resveratrol reverses Doxorubicin resistance by inhibiting epithelial-mesenchymal transition (EMT) through modulating PTEN/Akt signaling pathway in gastric cancer.

    PubMed

    Xu, Jiahui; Liu, Deying; Niu, Huilin; Zhu, Guifang; Xu, Yangwei; Ye, Danli; Li, Jian; Zhang, Qingling

    2017-01-26

    Gastric cancer is one of the major causes of cancer-related mortality worldwide. Most of patients presenting with inoperable gastric cancers rely on systemic chemotherapy for prolongation of survival. Doxorubicin (DOX) is one of the important agents against gastric cancer. Acquired DOX-resistance severely impedes the chemotherapeutic effect, invariably leading to poor prognosis. Resveratrol (RES) as a kind of phytoalexin has demonstrated anti-tumor functions in breast cancer and myeloid leukemia, but its function and mechanism are still unknown in gastric cancer treatment. CCK8 assay was used to detect the cytotoxicity of DOX and RES to gastric cancer cells. DOX-resistant subclone cell line (SGC7901/DOX) was derived from SGC7901 cells exposed to stepwise increasing concentrations of DOX treatment. We measured the migratory capabilities of SGC7901/DOX cells by Cell scratch test and Transwell assay. SGC7901/DOX cells were treated with DOX, RES, neither or both. Then we analyzed cell survival by CCK8 assay, colony formation by Colony-forming assay, cell apoptosis by Annexin-V-FITC and PI dual staining assay and cell migration by Cell scratch test and Transwell assay. Western blotting was conducted to detect the protein expressions of PTEN/Akt signaling pathway and EMT-related markers. Immunofluorescence was performed to confirm the EMT-related markers expressions. The xenograft model was used to assess the effect of DOX and RES in vivo. The key molecules associated with proliferation, apoptosis and EMT were evaluated by immunohistochemistry in tumor specimens. SGC7901/DOX cells acquired drug resistance and enhancive migratory capability. RES enabled SGC7901/DOX cells to regain DOX sensitivity, mitigated the aggressive biological features, promoted cell apoptosis in vitro and inhibited tumor growth in vivo. Mechanistic studies revealed that SGC7901/DOX cells underwent epithelial-mesenchymal transition (EMT) which was induced by Akt activation, and through activating

  8. [Effects of alginate/collagen scaffold on cell proliferation and differentiation of human adipose-derived mesenchymal stem cells].

    PubMed

    Cheng, W; Han, X P; Mou, S L; Yang, F; Liu, L P

    2017-04-09

    Objective: To build scaffold materials with different concentrations of alginate and collagen, and to observe the effects of alginate/collagen ratio on the proliferation of human adipose-derived mesenchymal stem cells (hAMSC) and osteogenic differentiation. The optimal concentration of alginate/collagen will be chosen for constructing hydrogel that will be used for bone tissue engineering. Methods: Soluble hydrogel scaffold materials containing alginate/collagen were prepared, and the following groups were established based on different alginate/collagen ratio: 4∶1 (group A), 2∶1 (group B), and 1∶1 (group C). Cell proliferation on the material surface was observed using the cell counting kit-8 (CCK-8) assay, while cell viability in each material group were observed using live/dead staining. Quantitative real-time PCR(qPCR) was used to measure the differential expression of osteogenesis-related genes on and in the materials. Immunofluorescence staining was used to measure the differential gene expression of osteogenesis-related proteins in each group. Results: The results from the CCK-8 assay showed increasing cell proliferation rate on the lyophilized hydrogel material surface as the collagen concentration increased, and the highest cell proliferation was observed in group C. Live/dead staining assay indicated that cells were able to proliferate in all three types of hydrogel materials, and the highest cell viability was found in material from group B ([87.50±2.65]%). qPCR showed that the expression of osteogenesis-related genes in group C was the highest, among the three groups, while the expression of osteocalcin in group B was significantly higher than those in the other two groups (P<0.05). Immunofluorescence staining was carried out for osteocalcin on and in the hydrogel material and the results were consistent with that of qPCR. Conclusions: The alginate/collagen scaffold materials did not show adverse effects on the cell proliferation of hAMSC and

  9. B7-H3 silencing inhibits tumor progression of mantle cell lymphoma and enhances chemosensitivity.

    PubMed

    Zhang, Wei; Wang, Yanfang; Wang, Jing; Dong, Fei; Zhu, Mingxia; Wan, Wenli; Li, Haishen; Wu, Feifei; Yan, Xinxing; Ke, Xiaoyan

    2015-01-01

    B7-H3 (CD276), known as a member of B7 immunoregulatory family, is a type I transmembrane glycoprotein aberrantly expressed in numerous types of cancer and associated with poor prognosis. However, the role of B7-H3 in oncogenesis and chemosensitivity of mantle cell lymphoma (MCL) remains unknown. We determined the effects of downregulating B7-H3 expression on tumor progression and the sensitivity of chemotherapeutic drug in mantle cell lymphoma. B7-H3 knockdown was performed using lentivirus transduction in the Maver and Z138 mantle cell lymphoma cell lines, respectively. The effects of B7-H3 on cell proliferation, cycle, migration and invasion were investigated by CCK-8 assay, methyl cellulose colony forming assay, PI staining, and Transwell assays in vitro. By establishing Maver and Z138 xenograft models, the effects of B7-H3 on tumorigenicity were observed, and Ki-67 and PCNA was detected by immunohistochemistry. The downregulation of B7-H3 significantly decreased tumor proliferation in MCL in vitro and in vivo. In the B7-H3 knockdown groups of Maver and Z138 xenograft models, the mean inhibition rate of tumor growth was 59.1 and 65.0% (p=0.010 and 0.003), and the expression of both Ki-67 and PCNA were significantly lower, respectively. After B7-H3 silencing, the cell cycles of Maver and Z138 were both arrested at G0/G1 phase, and the cell migration rates and invasion capacity were decreased as well. Moreover, the impacts of B7-H3 RNAi on the antitumor effect of chemotherapy drugs were determined with CCK-8 and Annexin V-FITC/PI assays in vitro and with xenograft models in vivo. The silencing of B7-H3 increased the sensitivity of Maver and Z138 cells to rituximab and bendamustine and enhanced the drug-induced apoptosis, respectively. Our study demonstrates for the first time that B7-H3 promotes mantle cell lymphoma progression and B7-H3 knockdown significantly enhances the chemosensitivity. This may provide a new therapeutic approach to mantle cell lymphoma.

  10. Uncovering potential of Indonesian medicinal plants on glucose uptake enhancement and lipid suppression in 3T3-L1 adipocytes.

    PubMed

    Lahrita, Lucy; Kato, Eisuke; Kawabata, Jun

    2015-06-20

    As obesity is a key factor in the development of type 2 diabetes, lowering lipid accumulation in adipose tissues is as important as increasing insulin sensitivity in diabetic patients. The selected plant extracts used in this screen have been traditionally used in Indonesian medicine for the treatment of diabetes and its complications. To investigate the ability of the selected plants to both increase insulin sensitivity through the enhancement of glucose uptake after insulin induction in adipocytes and suppress lipid production in the same target cells. Dried Indonesian medicinal plants were extracted with 50% (v/v) aq. methanol. The extracts were dissolved in 50% DMSO when tested in 3T3-L1 adipocytes. The screening platform consists of insulin-induced glucose uptake, lipid accumulation, and cell viability. Initially, an enzymatic fluorescence assay was designed to demonstrate the enhancement of 2-deoxyglucose (2-DG) uptake after insulin induction. Different concentrations of the extracts that enhanced glucose uptake were subjected to lipid accumulation assay using Oil Red O staining. Potential extracts based on lipid suppression were subsequently assessed by CCK-8 cell viability assay to distinguish lipid reduction activity from cytotoxicity. Out of 59 plants, 13 plants demonstrated their ability to increase glucose uptake in 3T3-L1 adipocytes after insulin induction, and 4 of these plants' extracts suppressed lipid production of the cells. The CCK-8 assay results of those 4 plant extracts suggest that the lipid inhibition activity of Eurycoma longifolia Jack (root) and Piper nigrum L. (fruits) extracts is not attributed to their cytotoxicity in the adipose cells. Both of the plant extracts increased glucose uptake by more than 200% at 50 μg/mL and suppressed lipid accumulation in a concentration-dependent manner. Screening of selected Indonesian medicinal plants has uncovered the potentials of E. longifolia Jack (root) and P. nigrum L. (fruits) with dual active

  11. Sauchinone augments cardiomyocyte viability by enhancing autophagy proteins -PI3K, ERK(1/2), AMPK and Beclin-1 during early ischemia-reperfusion injury in vitro

    PubMed Central

    Thapalia, Bisharad Anil; Zhou, Zhen; Lin, Xianhe

    2016-01-01

    Background. Sauchinone has proved its anti-oxidant and anti-inflammatory properties in various animal tissues. This study sought to illustrate its regulatory nature on autophagy associated proteins (PI3K, ERK1/2, AMPK, and Beclin-1) during early cardiomyocyte ischemia and subsequent reperfusion. Methods. Cultured cardiomyocytes were subjected to simulated Ischemia/reperfusion with and without Sauchinone pretreatment and also in the presence of autophagy inhibitor (3-MA). Colorimetric analysis of CCK-8, LDH antibody assay as well as Western blot analysis were performed to observe the expressions of LC3B (II) and Beclin-1 protein (markers of autophagy), autophagy proteins (PI3K, ERK1/2 and AMPK) and apoptotic proteins (Bax and Bcl-2) and the results were quantified into their grey values and subjected to statistical analysis. Results. Sauchinone demonstrated cell survival enhancing properties with increase in CCK-8 (SD = 0.553±0.012) and decrease in LDH (SD = 0.183±0.054) expressions, both of which were best observed at test dose of 20 µmol/L. At this dose, there was increment in cellular autophagy as demonstrated by peaking of autophagy markers LC3B-II (p<0.05) and Beclin-1 (p<0.05) with strong correlations (r = 0.99). Similarly, the autophagy proteins, compared to control and I/R model, also showed a significant increased level with PI3K (p<0.0001), total p-ERK1/2 (p<0.0001) and p-AMPKα (p<0.0001). Simultaneously, a decrease in expressions of pro-apoptotic molecules Bax (r = 0.989, p<0.0001) with increment of in the anti-apoptotic protein Bcl-2 (r = 0.996, p<0.0001) was observed. The observed effects on cell density, viability and autophagy was abrogated in presence of 3-MA. Conclusions. Sauchinone enhances cell survival by promoting autophagy and inhibiting apoptosis in cardiomyocytes during early stages of Ischemia/reperfusion injury. PMID:27508047

  12. Anticancer effect of salidroside on colon cancer through inhibiting JAK2/STAT3 signaling pathway

    PubMed Central

    Sun, Kuan-Xue; Xia, Hong-Wei; Xia, Rong-Long

    2015-01-01

    Salidroside is considered to have anti-tumor properties. We investigate its effects on colon carcinoma SW1116 cells. Cell viability was assessed by CCK-8. Propidium iodide (PI) staining was used to determine the cell cycle by flow cytometry. The migration and invasion were detected by Transwell. Western blot was used to detect the expression of STAT3 signal related proteins. As the result, high concentrations of salidroside (10, 20. 50 μg/ml) significantly inhibited proliferation of SW1116 cells in a parallelly, cell cycle arrest was increased at the G0/G1 phase after salidroside treatment. Furthermore, salidroside inhibited migration and invasion of SW1116 cells. Salidroside treatment decreased proteins expression of phosphorylation levels in JAK2/STAT3 signaling, while MMP-2 and MMP-9 proteins levels were decreased and protein expression of VEGF and VEGFR-2 were down-regulated. In Conclusion, salidroside inhibited proliferation, decreased the migration and invasion of SW1116 cells in JAK2/STAT3-dependent pathway, the specific mechanisms need further study. PMID:25755753

  13. Anticancer effect of salidroside on colon cancer through inhibiting JAK2/STAT3 signaling pathway.

    PubMed

    Sun, Kuan-Xue; Xia, Hong-Wei; Xia, Rong-Long

    2015-01-01

    Salidroside is considered to have anti-tumor properties. We investigate its effects on colon carcinoma SW1116 cells. Cell viability was assessed by CCK-8. Propidium iodide (PI) staining was used to determine the cell cycle by flow cytometry. The migration and invasion were detected by Transwell. Western blot was used to detect the expression of STAT3 signal related proteins. As the result, high concentrations of salidroside (10, 20. 50 μg/ml) significantly inhibited proliferation of SW1116 cells in a parallelly, cell cycle arrest was increased at the G0/G1 phase after salidroside treatment. Furthermore, salidroside inhibited migration and invasion of SW1116 cells. Salidroside treatment decreased proteins expression of phosphorylation levels in JAK2/STAT3 signaling, while MMP-2 and MMP-9 proteins levels were decreased and protein expression of VEGF and VEGFR-2 were down-regulated. In Conclusion, salidroside inhibited proliferation, decreased the migration and invasion of SW1116 cells in JAK2/STAT3-dependent pathway, the specific mechanisms need further study.

  14. Long Non-Coding RNA (lncRNA) Urothelial Carcinoma-Associated 1 (UCA1) Enhances Tamoxifen Resistance in Breast Cancer Cells via Inhibiting mTOR Signaling Pathway

    PubMed Central

    Wu, Chihua; Luo, Jing

    2016-01-01

    Background Long non-coding RNA (lncRNA) UCA1 is an oncogene in breast cancer. The purpose of this study was to investigate the role of UCA1 in tamoxifen resistance of estrogen receptor positive breast cancer cells. Material/Methods Tamoxifen sensitive MCF-7 cells were transfected for UCA1 overexpression, while tamoxifen resistant LCC2 and LCC9 cells were transfected with UCA siRNA for UCA1 knockdown. qRT-PCR was performed to analyze UCA1 expression. CCK-8 assay, immunofluorescence staining of cleaved caspase-9, and flow cytometric analysis of Annexin V/PI staining were used to assess tamoxifen sensitivity. Western blot analysis was performed to detect p-AKT and p-mTOR expression. Results LncRNA UCA1 was significantly upregulated in tamoxifen resistant breast cancer cells compared to tamoxifen sensitive cells. LCC2 and LCC9 cells transfected with UCA1 siRNA had significantly higher ratio of apoptosis after tamoxifen treatment. UCA1 siRNA significantly decreased the protein levels of p-AKT and p-mTOR in LCC2 and LCC9 cells. Enforced UCA1 expression substantially reduced tamoxifen induced apoptosis in MCF-7 cells, while rapamycin treatment abrogated the protective effect of UCA1. Conclusions UCA1 upregulation was associated with tamoxifen resistance in breast cancer. Mechanistically, UCA1 confers tamoxifen resistance to breast cancer cells partly via activating the mTOR signaling pathway. PMID:27765938

  15. Kaempferol inhibited VEGF and PGF expression and in vitro angiogenesis of HRECs under diabetic-like environment

    PubMed Central

    Xu, X.H.; Zhao, C.; Peng, Q.; Xie, P.; Liu, Q.H.

    2017-01-01

    Diabetic retinopathy (DR) is one of the common and specific microvascular complications of diabetes. This study aimed to investigate the anti-angiogenic effect of kaempferol and explore its underlying molecular mechanisms. The mRNA expression level of vascular endothelial growth factor (VEGF) and placenta growth factor (PGF) and the concentrations of secreted VEGF and PGF were measured by qTR-PCR and ELISA assay, respectively. Human retinal endothelial cells (HRECs) proliferation, migration, and sprouting were measured by CCK-8 and transwell, scratching wound, and tube formation assays, respectively. Protein levels were determined by western blot. High glucose (25 mM) increased the mRNA expression levels of VEGF and PGF as well as the concentrations of secreted VEGF and PGF in HRECs, which can be antagonized by kaempferol (25 µM). Kaempferol (5-25 µM) significantly suppressed cell proliferation, migration, migration distance and sprouting of HRECs under high glucose condition. The anti-angiogenic effect of kaempferol was mediated via downregulating the expression of PI3K and inhibiting the activation of Erk1/2, Src, and Akt1. This study indicates that kaempferol suppressed angiogenesis of HRECs via targeting VEGF and PGF to inhibit the activation of Src-Akt1-Erk1/2 signaling pathway. The results suggest that kaempferol may be a potential drug for better management of DR. PMID:28273207

  16. Alternating block polyurethanes based on PCL and PEG as potential nerve regeneration materials.

    PubMed

    Li, Guangyao; Li, Dandan; Niu, Yuqing; He, Tao; Chen, Kevin C; Xu, Kaitian

    2014-03-01

    Polyurethanes with regular and controlled block arrangement, i.e., alternating block polyurethanes (abbreviated as PUCL-alt-PEG) based on poly(ε-caprolactone) (PCL-diol) and poly(ethylene glycol) (PEG) was prepared via selectively coupling reaction between PCL-diol and diisocyanate end-capped PEG. Chemical structure, molecular weight, distribution, and thermal properties were systematically characterized by FTIR, (1)H NMR, GPC, DSC, and TGA. Hydrophilicity was studied by static contact angle of H2O and CH2I2. Film surface was observed by scanning electron microscope (SEM) and atomic force microscopy, and mechanical properties were assessed by universal test machine. Results show that alternating block polyurethanes give higher crystal degree, higher mechanical properties, and more hydrophilic and rougher (deep ravine) surface than their random counterpart, due to regular and controlled structure. Platelet adhesion illustrated that PUCL-alt-PEG has better hemocompatibility and the hemacompatibility was affected significantly by PEG content. Excellent hemocompatibility was obtained with high PEG content. CCK-8 assay and SEM observation revealed much better cell compatibility of fibroblast L929 and rat glial cells on the alternating block polyurethanes than that on random counterpart. Alternating block polyurethane PUC20-a-E4 with optimized composition, mechanical, surface properties, hemacompatibility, and highest cell growth and proliferation was achieved for potential use in nerve regeneration.

  17. Allyl Isothiocyanate Inhibits the Proliferation of Renal Carcinoma Cell Line GRC-1 by Inducing an Imbalance Between Bcl2 and Bax

    PubMed Central

    Jiang, Zhongyong; Liu, Xi; Chang, Kai; Liu, Xia; Xiong, Jie

    2016-01-01

    Background Because of the insensitivity of renal cell carcinoma (RCC) to both chemotherapy and radiotherapy, surgery remains the primary approach for anticancer treatment. However, patients who do not receive timely diagnoses may not be suitable for surgery, especially in the late phase of tumor development. Thus, the discovery of novel effective treatment is of great importance. Allyl isothiocyanate (AITC) can inhibit the proliferation and induce apoptosis in many cancer cells. In this paper, we report on an in vitro study to determine the effect of AITC on proliferation and apoptosis of RCC line GRC-1. Material/Methods CCK8 assay was used to detect cell proliferation under gradient concentrations of AITC. Flow cytometry was employed to evaluate cell apoptosis. Real-time fluorescent polymerase chain reaction quantified mRNA levels of Bax and Bcl-2 genes. Western blotting was further employed for protein expression assay. Results AITC inhibited GRC-1 cell proliferation and induced cell apoptosis in a dose-dependent manner; it also elevated Bax while suppressing Bcl-2 gene expression at both mRNA and protein levels. In general, increasing concentration of AITC decreased Bcl-2/Bax ratio. Conclusions The inhibitory effect of AITC on GRC-1 cells is exerted via cell apoptosis, in which the imbalance of Bcl-2/Bax plays a significant role. PMID:27834342

  18. Preparation and application of a novel molecularly imprinted solid-phase microextraction monolith for selective enrichment of cholecystokinin neuropeptides in human cerebrospinal fluid.

    PubMed

    Ji, Xiang; Li, Dan; Li, Hua

    2015-08-01

    A novel molecularly imprinted polymer (MIP) monolith for highly selective extraction of cholecystokinin (CCK) neuropeptides was prepared in a micropipette tip. The MIPs were synthesized by epitope imprinting technique and the polymerization conditions were investigated and optimized. The synthesized MIPs were characterized by infrared spectroscopy, elemental analyzer and scanning electron microscope. A molecularly imprinted solid-phase microextraction (MI-μ-SPE) method was developed for the extraction of CCK neuropeptides in aqueous solutions. The parameters affecting MI-μ-SPE were optimized. The results indicated that this MIP monolith exhibited specific recognition capability and high enrichment efficiency for CCK neuropeptides. In addition, it showed excellent reusability. This MIP monolith was used for desalting and enrichment of CCK4, CCK5 and CCK8 from human cerebrospinal fluid prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, and the results show that this MIP monolith can be a useful tool for effective purification and highly selective enrichment of multiple homologous CCK neuropeptides in cerebrospinal fluid simultaneously. By employing MI-μ-SPE combined with HPLC-ESI-MS/MS analysis, endogenous CCK4 in human cerebrospinal fluid was quantified.

  19. Silencing Livin induces apoptotic and autophagic cell death, increasing chemotherapeutic sensitivity to cisplatin of renal carcinoma cells.

    PubMed

    Wang, Zhiyang; Liu, Shuai; Ding, Kejia; Ding, Sentai; Li, Chensheng; Lu, Jiaju; Gao, Dexuan; Zhang, Tong; Bi, Dongbin

    2016-11-01

    Renal cell carcinoma (RCC) accounts for 3 % of all adult malignancies and is the most lethal urological cancer. Livin is a member of the inhibitor of apoptosis protein (IAP) family, which is associated with tumor resistance to radiotherapy and chemotherapy. Clinical data also showed that patients with high tumor grades and stages have higher expression levels of Livin in RCC cells. Autophagy is a survival mechanism activated in response to nutrient deprivation. A possible role of Livin in the autophagy of RCC cells has not been investigated; therefore, this pioneer study was carried out. Livin was silenced in RCC cells (slow virus infection [SVI]-shLivin cells) by lentiviral transfection. Then, mRNA and protein expression levels in the transfected cells were assessed by quantitative fluorescence PCR and Western blotting, respectively. In addition, acridine orange staining and electron microscopy were used to assess autophagy in SVI-shLivin cells. The cisplatin IC50 values for RCC cells were measured by the CCK8 assay. Potent antitumor activities were observed in xenograft mouse models generated with Livin-silenced RCC cells in terms of delayed tumor onset and suppressed tumor growth. These results suggested that Livin silencing could increase the chemotherapeutic sensitivity of RCC cells to cisplatin and induce autophagic cell death. A possible mechanism of Bcl-2 and Akt pathway involvement was discussed specifically in this study. Overall, Livin silencing induces apoptotic and autophagic cell death and increases chemotherapeutic sensitivity of RCC cells to cisplatin.

  20. Downregulation of cancer stem cell properties via mTOR signaling pathway inhibition by rapamycin in nasopharyngeal carcinoma

    PubMed Central

    YANG, CHUNGUANG; ZHANG, YUE; ZHANG, YU; ZHANG, ZIHENG; PENG, JIANHUA; LI, ZHI; HAN, LIANG; YOU, QUANJIE; CHEN, XIAOYU; RAO, XINGWANG; ZHU, YI; LIAO, ZHISU

    2015-01-01

    Rapamycin, a mammalian target of rapamycin (mTOR) signaling inhibitor, inhibits cancer cell proliferation and tumor formation, including in nasopharyngeal carcinoma (NPC), which we proved in a previous study. However, whether rapamycin affects cancer stem cells (CSCs) is unclear. In examining samples of NPCs, we found regions of CD44-positive cancer cells co-expressing the stem cell biomarker OCT4, suggesting the presence of CSCs. Following this, we used double-label immunohistochemistry to identify whether the mTOR signaling pathway was activated in CD44-positive CSCs in NPCs. We used a CCK-8 assay and western blotting to explore whether the stem cell biomarkers CD44 and SOX2 and the invasion protein MMP-2 could be suppressed by treatment with rapamycin in cultured primary NPC cells and secondary tumors in BALB/c nude mice. Interestingly, we found that rapamycin inhibited mTOR signaling in addition to simultaneously downregulating the expression of CD44, SOX2 and MMP-2 and that it affected cell growth and tumor size and weight both in vitro and in vivo. Collectively, we confirmed for the first time that CSC properties are reduced and invasion potential is restrained in response to mTOR signaling inhibition in NPC. This evidence indicates that the targeted inhibition of CSC properties may provide a novel strategy to treat cancer. PMID:26202311

  1. Astragalus polysaccharide induces the apoptosis of human hepatocellular carcinoma cells by decreasing the expression of Notch1.

    PubMed

    Huang, Wen-Hai; Liao, Wei-Rong; Sun, Rong-Xun

    2016-08-01

    Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer death worldwide. Astragalus polysaccharide (APS), the primary active component extracted from a traditional Chinese medicinal herb Astragalus membranaceus, has been proved to exert a marked inhibitory effect on a number of types of human solid tumors. In the present study, we aimed to examine the effects of APS on the survival of the HCC cell line H22 and to elucidate the underlying regulatory mechanisms responsible for these effects. Our results revealed that the mRNA and protein expression of Notch1 was significantly upregulated in the HCC tissues compared with that in the normal tissues. APS decreased cell viability and induced the apoptosis of HCC cells in a concentration-dependent manner, which were evaluated using a cell counting kit-8 (CCK-8) assay and flow cytometric analysis, respectively. Furthermore, APS regulated the expression of apoptosis-related genes (Bcl-2 and BAX) and proteases (caspase-3 and -8). Mechanically, Notch1 expression was found to be suppressed in HCC cells, and further analysis indicated that Notch1 knockdown by siRNA significantly reduced cell viability, suppressed the metastatic capacity and enhanced the apoptosis of HCC cells. Taken together, these findings suggest that Notch1 may be a potential therapeutic target for the treatment of HCC.

  2. Astragalus saponins affect proliferation, invasion and apoptosis of gastric cancer BGC-823 cells

    PubMed Central

    2013-01-01

    Background Astragalus memebranaceus is a traditional Chinese herbal medicine used in treatment of common cold, diarrhea, fatigue, anorexia and cardiac diseases. Recently, there are growing evidences that Astragalus extract may be a potential anti-tumorigenic agent. Some research showed that the total saponins obtained from Astragalus membranaceus possess significant antitumorigenic activity. Gastric cancer is one of the most frequent cancers in the world, almost two-thirds of gastric cancer cases and deaths occur in less developed regions. But the effect of Astragalus membranaceus on proliferation, invasion and apoptosis of gastric cancer BGC-823 cells remains unclear. Methods Astragalus saponins were extracted. Cells proliferation was determined by CCK-8 assay. Cell cycle and apoptosis were detected by the flow cytometry. Boyden chamber was used to evaluate the invasion and metastasis capabilities of BGC-823 cells. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. Results The results demonstrated that total Astragalus saponins could inhibit human gastric cancer cell growth both in vitro and in vivo, in additional, Astragalus saponins deceased the invasion ability and induced the apoptosis of gastric cancer BGC-823 cells. Conclusions Total Astragalus saponins inhibited human gastric cancer cell growth, decreased the invasion ability and induced the apoptosis. This suggested the possibility of further developing Astragalus as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent in gastric cancer therapy. PMID:24152941

  3. Astragalus saponins affect proliferation, invasion and apoptosis of gastric cancer BGC-823 cells.

    PubMed

    Wang, Tao; Xuan, Xiaoyan; Li, Min; Gao, Ping; Zheng, Yuling; Zang, Wenqiao; Zhao, Guoqiang

    2013-10-24

    Astragalus memebranaceus is a traditional Chinese herbal medicine used in treatment of common cold, diarrhea, fatigue, anorexia and cardiac diseases. Recently, there are growing evidences that Astragalus extract may be a potential anti-tumorigenic agent. Some research showed that the total saponins obtained from Astragalus membranaceus possess significant antitumorigenic activity. Gastric cancer is one of the most frequent cancers in the world, almost two-thirds of gastric cancer cases and deaths occur in less developed regions. But the effect of Astragalus membranaceus on proliferation, invasion and apoptosis of gastric cancer BGC-823 cells remains unclear. Astragalus saponins were extracted. Cells proliferation was determined by CCK-8 assay. Cell cycle and apoptosis were detected by the flow cytometry. Boyden chamber was used to evaluate the invasion and metastasis capabilities of BGC-823 cells. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. The results demonstrated that total Astragalus saponins could inhibit human gastric cancer cell growth both in vitro and in vivo, in additional, Astragalus saponins deceased the invasion ability and induced the apoptosis of gastric cancer BGC-823 cells. Total Astragalus saponins inhibited human gastric cancer cell growth, decreased the invasion ability and induced the apoptosis. This suggested the possibility of further developing Astragalus as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent in gastric cancer therapy.

  4. Protective effects of quercetin and taraxasterol against H2O2-induced human umbilical vein endothelial cell injury in vitro

    PubMed Central

    YANG, DONGWEI; LIU, XINYE; LIU, MIN; CHI, HAO; LIU, JIRONG; HAN, HUAMIN

    2015-01-01

    Due to the association between inflammation and endothelial dysfunction in atherosclerosis, the blockage of the inflammatory process that occurs on the endothelial cells may be a useful way of preventing atherosclerosis. In the present study, human umbilical vein endothelial cells (HUVECs) were used to investigate the protective effects of quercetin and taraxasterol against H2O2-induced oxidative damage and inflammation. HUVECs were pretreated with quercetin or taraxasterol at concentrations ranging between 0 and 210 µM for 12 h, prior to being administered different concentrations of H2O2 for 4 h. Cell viability and levels of apoptosis were assessed through cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling assays, respectively, to determine the injury to the HUVECs. The viability loss in the H2O2-induced HUVECs was markedly restored in a concentration-dependent manner by pretreatment with quercetin or taraxasterol. This effect was accompanied by significantly decreased expression of vascular cell adhesion molecule 1 (VCAM-1) and cluster of differentiation (CD)80 for taraxasterol and that of CD80 for quercetin. In conclusion, the present study showed the protective effects of quercetin and taraxasterol against cell injury and inflammation in HUVECs and indicated that the effects were mediated via the downregulation of VCAM-1 and CD80 expression. This study has therefore served as a preliminary investigation on the anti-atherosclerotic and cardiovascular protective effects of quercetin and taraxasterol as dietary supplements. PMID:26622474

  5. Treatment of steroid-induced osteonecrosis of the femoral head using porous Se@SiO2 nanocomposites to suppress reactive oxygen species

    NASA Astrophysics Data System (ADS)

    Deng, Guoying; Niu, Kerun; Zhou, Feng; Li, Buxiao; Kang, Yingjie; Liu, Xijian; Hu, Junqing; Li, Bo; Wang, Qiugen; Yi, Chengqing; Wang, Qian

    2017-03-01

    Reducing oxidative stress (ROS) have been demonstrated effective for steroid-induced osteonecrosis of the femoral head (steroid-induced ONFH). Selenium (Se) plays an important role in suppressing oxidative stress and has huge potential in ONFH treatments. However the Se has a narrow margin between beneficial and toxic effects which make it hard for therapy use in vivo. In order to make the deficiency up, a control release of Se (Se@SiO2) were realized by nanotechnology modification. Porous Se@SiO2 nanocomposites have favorable biocompatibility and can reduced the ROS damage effectively. In vitro, the cck-8 analysis, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) stain and flow cytometry analysis showed rare negative influence by porous Se@SiO2 nanocomposites but significantly protective effect against H2O2 by reducing ROS level (detected by DCFH-DA). In vivo, the biosafety of porous Se@SiO2 nanocomposites were confirmed by the serum biochemistry, the ROS level in serum were significantly reduced and the curative effect were confirmed by Micro CT scan, serum Elisa assay (inflammatory factors), Western blotting (quantitative measurement of ONFH) and HE staining. It is expected that the porous Se@SiO2 nanocomposites may prevent steroid-induced ONFH by reducing oxidative stress.

  6. Sustained PDGF-BB release from PHBHHx loaded nanoparticles in 3D hydrogel/stem cell model.

    PubMed

    Dong, Cui-Ling; Webb, William R; Peng, Qiang; Tang, James Z; Forsyth, Nicholas R; Chen, Guo-Qiang; El Haj, Alicia J

    2015-01-01

    This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering. © 2014 Wiley Periodicals, Inc.

  7. Nuclear transcription factor Nrf2 suppresses prostate cancer cells growth and migration through upregulating ferroportin.

    PubMed

    Xue, Dong; Zhou, Cuixing; Shi, Yunbo; Lu, Hao; Xu, Renfang; He, Xiaozhou

    2016-11-29

    VTo investigate the effect of nuclear transcription factor Nrf2 on the transcription of Ferroportin (FPN) in prostate cancer cells, and the regulation mechanisms of FPN on cell viability, migration and apoptosis of prostate cancer cells.Empty vectors, pEGFPC1-Nrf2, pEGFPC1-FPN, Si-FPN and Si-Nrf2 were transfected into prostate cancer cell line PC3. The expression of mRNA and protein were measured by real time-PCR (RT-PCR) and western blot. Cell viability, migration, cycle and apoptosis were tested by CCK-8 assay, wound healing and flow cytometry, respectively. The interaction between FPN and Nrf2 was confirmed by chromatin immunoprecipitation (CHIP) assay.The viability, migration and mitosis of PC3 cells could be repressed by over-expressed FPN, with decreased intracellular ferritin. The CHIP assay demonstrated that Nrf2 is one transcription factor of FPN and promotes its transcription. With the increase of Nrf2 in PC3 cells, the viability, migration ability and concentration of ferritin were suppressed, while the apoptosis rate was increased. The above effects were counteracted by down-regulating FPN.FPN could inhibit the prostate cancer cell viability, migration and mitosis, which is also related to a decrease of intracellular ferritin content. In conclusion, Nrf2 suppresses prostate cancer cells viability, migration, and mitosis through upregulating FPN.

  8. Effects of anodic titanium dioxide nanotubes of different diameters on macrophage secretion and expression of cytokines and chemokines.

    PubMed

    Lü, W L; Wang, N; Gao, P; Li, C Y; Zhao, H S; Zhang, Z T

    2015-02-01

    To investigate effects of TiO2 nanotubes of different diameters on J744A.1 macrophage behaviour, secretion and expression of pro-inflammatory cytokines and chemokines. Macrophage-like J744A.1 cells were cultured on three types of Ti surface: mechanically polished titanium plus 30 and 80 nm TiO2 nanotube surfaces, for 4, 24 and 48 h. Macrophage adhesion and proliferation were assessed using CCK-8 assay. Levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokines (MCP-1 and MIP-1α) secreted into the supernatant were measured using the Cytometric Bead Arrays kit. TNF-α, MCP-1 and MIP-1α gene expression were quantitatively analysed by real-time PCR. These show that TiO2 nanotube surfaces, especially of 80 nm TiO2 nanotube, benefited macrophage adhesion and proliferation, and reduced protein secretion and mRNA expression of TNF-α, MCP-1 and MIP-1α. IL-1β and IL-6 were undetectable on all the surfaces at all times. TiO2 nanotube surfaces, especially of 80 nm TiO2 nanotube, reduced inflammatory response in vitro, which might be part of a basis for rapid osseointegration in implants with TiO2 nanotube surfaces in animal studies. © 2014 John Wiley & Sons Ltd.

  9. Controlling mesenchymal stem cells differentiate into contractile smooth muscle cells on a TiO2 micro/nano interface: Towards benign pericytes environment for endothelialization.

    PubMed

    Li, Jingan; Qin, Wei; Zhang, Kun; Wu, Feng; Yang, Ping; He, Zikun; Zhao, Ansha; Huang, Nan

    2016-09-01

    Building healthy and oriented smooth muscle cells (SMCs) environment is an effective method for improving the surface endothelialization of the cardiovascular implants. However, a long-term and stable source of SMCs for implantation without immune rejection and inflammation has not been solved, and mesenchymal stem cells (MSCs) differentiation may be a good choice. In this work, two types of TiO2 micro/nano interfaces were fabricated on titanium surface by photolithography and anodic oxidation. These TiO2 micro/nano interfaces were used to regulate the differentiation of the MSCs. The X-ray diffraction (XRD) detection showed that the TiO2 micro/nano interfaces possessed the anatase crystal structure, suggesting good cytocompatibility. The CCK-8 results indicated the TiO2 micro/nano interfaces improved MSC proliferation, further immunofluorescence staining and calculation of the cell morphology index proved the micro/nano surfaces also elongated MSCs and regulated MSCs oriented growth. The specific staining of α-SMA, CNN-1, vWF, CD44 and CD133 markers revealed that the micro/nano surfaces induced MSCs differentiation to contractile SMCs, and the endothelial cells (ECs) culture experiment indicated that the MSCs induced by micro/nano interfaces contributed to the ECs attachment and proliferation. This method will be further studied and applied for the surface modification of the cardiovascular implants.

  10. DNMT1 regulates human endometrial carcinoma cell proliferation

    PubMed Central

    Wang, Xinjing; Li, Bilan

    2017-01-01

    Endometrial carcinoma (EC) is the most common gynecologic malignancy, but the molecular events involved in the development and progression of EC remain unclear. This study aimed to investigate the role of DNA methyltransferase 1 (DNMT1), a member of DNA methyltransferases, in EC. AN3CA cells were transfected with DNMT1 siRNA. The proliferation, cell cycle, and apoptosis of AN3CA cells were evaluated by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The expression of related genes was detected by polymerase chain reaction and Western blot analysis. Knockdown of DNMT1 inhibited the proliferation, induced apoptosis, and G0/G1 phase arrest of AN3CA cells. Furthermore, knockdown of DNMT1 upregulated the expression of nuclear factor kappa-B-inhibitor alpha (NF-κBIA) and Bax and downregulated the expression of Bcl-2 and CCND1/2 in AN3CA cells. In conclusion, this study provides the first evidence that knockdown of DNMT1 affects the expression of cell cycle- and apoptosis-associated proteins in EC cells, suggesting the potential of DNMT1 in EC therapy. PMID:28408839

  11. Effect of Melatonin on the Extracellular-Regulated Kinase Signal Pathway Activation and Human Osteoblastic Cell Line hFOB 1.19 Proliferation

    PubMed Central

    Xiong, Xiao-Chuan; Zhu, Yue; Ge, Rui; Liu, Li-Feng; Yuan, Wei

    2015-01-01

    It has been shown that melatonin may affect bone metabolism. However, it is controversial whether melatonin could promote osteoblast proliferation, and the precise molecular mechanism of melatonin on osteoblast proliferation is still obscure. In this study, the results of the CCK-8 assay showed that melatonin significantly promoted human osteoblastic cell line hFOB 1.19 cell proliferation at 1, 2.5, 5, 10, 25, 50 and 100 µM concentrations for 24 h, but there were no significant differences among the groups. Western blot demonstrated that 10 µM melatonin significantly promoted ERK1/2 phosphorylation. Furthermore, we also detected the phosphorylation of c-Raf, MEK1/2, p90RSK and MSK1, and all of them increased with 10 µM melatonin. U0126 (a selective inhibitor of MEK that disrupts downstream activation of ERK1/2) downregulated the phosphorylation of ERK1/2, p90RSK and MSK1. U0126 also attenuated the proliferation of osteoblasts stimulated by melatonin. In conclusion, this study for the first time indicates that melatonin (10 nM–100 µM) promotes the proliferation of a human osteoblastic cell line hFOB 1.19 through activation of c-Raf, MEK1/2, ERK1/2, p90RSK and MSK1. PMID:25961946

  12. Systemic cholecystokinin amplifies vago-vagal reflex responses recorded in vagal motor neurones.

    PubMed

    Viard, Edouard; Rogers, Richard C; Hermann, Gerlinda E

    2012-02-01

    Cholecystokinin (CCK) is a potent regulator of visceral functions as a consequence of its actions on vago-vagal reflex circuit elements. This paper addresses three current controversies regarding the role of CCK to control gastric function via vago-vagal reflexes. Specifically: (a) whether CNS vs. peripheral (vagal afferent) receptors are dominant, (b) whether the long (58) vs. short (8) isoform is more potent and (c) whether nutritional status impacts the gain or even the direction of vago-vagal reflexes. Our in vivo recordings of physiologically identified gastric vagal motor neurones (gastric-DMN) involved in the gastric accommodation reflex (GAR) show unequivocally that: (a) receptors in the coeliac-portal circulation are more sensitive in amplifying gastric vagal reflexes; (b) in the periphery, CCK8 is more potent than CCK58; and (c) the nutritional status has a marginal effect on gastric reflex control. While the GAR reflex is more sensitive in the fasted rat, CCK amplifies this sensitivity. Thus, our results are in stark contrast to recent reports which have suggested that vago-vagal reflexes are inverted by the metabolic status of the animal and that this inversion could be mediated by CCK within the CNS.

  13. Effects of the extract of Ginkgo biloba on the differentiation of bone marrow mesenchymal stem cells in vitro

    PubMed Central

    Wu, Zhe; Zhang, Jiadi; Gu, Xu; Zhang, Xiaoxiao; Shi, Shuman; Liu, Chang

    2016-01-01

    The balance of osteogenesis and adipogenesis in bone marrow mesenchymal stem cells (BMSCs) is disrupted in osteoporosis. This study was designed to investigate the effects of extract of Ginkgo biloba (EGB) on proliferation, osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells in vitro. The effect of EGB on proliferation was evaluated by CCK-8 assay and flow cytometry. Osteogenic differentiation was evaluated by Alizarin Red S staining and Alkaline phosphatase assay. Adipogenic differentiation was evaluated by Oil Red O staining. Quantitative real-time polymerase chain reaction (Real-time PCR) was used to detect the expression of osteogenic specific genes (BMP-2, Runx2 and Colla1) and adipogenic specific genes (ap2, PPARγ). EGB did not significantly affect proliferation of BMSCs. However, it increased the calcium accumulation and significantly promoted the activity of alkaline phosphatase, especially when the concentration of EGB reached 150 µg/mL. EGB dose-dependently inhibited the adipogenic ability of BMSCs. The osteogenic-related genes (BMP-2, Runx2, Colla1) were overexpressed while the expression of genes involved in adipogenesis, such as PPAR-γ and ap2, was decreasing with the increase of EGB concentration. Our data proves that EGB inhibited adipocyte differentiation and enhanced osteogenic differentiation in BMSCs, but had no effect on the proliferation of BMSCs. PMID:27508023

  14. Up-regulation of TDAG51 is a dependent factor of LPS-induced RAW264.7 macrophages proliferation and cell cycle progression.

    PubMed

    Jiao, Han-Wei; Jia, Xiao-Xiao; Zhao, Tian-Jing; Rong, Hui; Zhang, Jia-Ning; Cheng, Ying; Zhu, Hua-Pei; Xu, Kai-Lian; Guo, Shi-Yu; Shi, Qiao-Yun; Zhang, Hui; Wang, Feng-Yang; Chen, Chuang-Fu; Du, Li

    2016-01-01

    As a component of the outer membrane in Gram-negative bacteria, lipopolysaccharide (LPS)-induced proliferation and cell cycle progression of monocytes/macrophages. It has been suggested that the proapoptotic T-cell death-associated gene 51 (TDAG51) might be associated with cell proliferation and cell cycle progression; however, its role in the interaction between LPS and macrophages remains unclear. We attempted to elucidate the role(s) of TDAG51 played in the interaction between LPS and macrophages. We investigated TDAG51 expression in RAW264.7 cells stimulated with LPS and examined the effects of RNA interference-mediated TDAG51 down-regulation. We used CCK-8 assay and flow cytometry analysis to evaluate the interaction between TDAG51 and LPS-induced proliferation and cell cycle progression in RAW264.7 cells. Our findings indicate that TDAG51 is up-regulated in LPS-stimulated RAW264.7 cells, the TDAG51 siRNA effectively reduced TDAG51 protein up-regulation following LPS stimulation in RAW264.7 cells, the significant changes of the proliferation and cell cycle progression of RAW264.7 cells in TDAG51 Knockdown RAW264.7 cells treated with LPS were observed. These findings suggested that TDAG51 up-regulation is a dependent event during LPS-mediated proliferation and cell cycle progression, and which increase our understanding of the interaction mechanism between LPS and macrophages.

  15. Calreticulin down-regulation inhibits the cell growth, invasion and cell cycle progression of human hepatocellular carcinoma cells.

    PubMed

    Feng, Ruo; Ye, Jianwen; Zhou, Chuang; Qi, Lei; Fu, Zhe; Yan, Bing; Liang, Zhiwei; Li, Renfeng; Zhai, Wenlong

    2015-08-27

    Hepatocellular carcinoma (HCC) is one of the most frequent cancers in the world. Calreticulin(CRT) is aberrantly overexpressed in many human cancer cells. The function of CRT in HCC cells remains unclear. We attempted to investigate the effects and the underlying mechanisms of CRT down-regulation on HCC cell growth, apoptosis, cell cycle progression and invasion. To investigate the function of CRT in HCC cells, small interfering RNA (siRNA) was used to knock down the expression of CRT in SMMC7721 and HepG2 HCC cells. CRT expression was examined by Western blot and immunofluorescence. Cell proliferation was detected by CCK-8 assay. Cell cycle and apoptosis were measured by the flow cytometry. The invasion capability was assessed by transwell assay. The phosphorylation level of Akt was evaluated by Western blot. Compared with human hepatic cells L02, CRT was apparently up-regulated in SMMC7721, HepG2 and Huh7 HCC cells. Down-regulation of CRT expression effectively inhibited HCC cell growth and invasion. CRT knockdown induced cell cycle arrest and the apoptosis in SMMC7721 and HepG2 cells. Furthermore, down-regulation of CRT expression significantly decreased the Akt phosphorylation. CRT was aberrantly over-expressed in HCC cell lines. CRT over-expression contributes greatly to HCC malignant behavior, likely via PI3K/Akt pathway. CRT could serve as a potential biomarker and therapeutic target for hepatocellular carcinoma.

  16. Treatment of steroid-induced osteonecrosis of the femoral head using porous Se@SiO2 nanocomposites to suppress reactive oxygen species

    PubMed Central

    Deng, Guoying; Niu, Kerun; Zhou, Feng; Li, Buxiao; Kang, Yingjie; Liu, Xijian; Hu, Junqing; Li, Bo; Wang, Qiugen; Yi, Chengqing; Wang, Qian

    2017-01-01

    Reducing oxidative stress (ROS) have been demonstrated effective for steroid-induced osteonecrosis of the femoral head (steroid-induced ONFH). Selenium (Se) plays an important role in suppressing oxidative stress and has huge potential in ONFH treatments. However the Se has a narrow margin between beneficial and toxic effects which make it hard for therapy use in vivo. In order to make the deficiency up, a control release of Se (Se@SiO2) were realized by nanotechnology modification. Porous Se@SiO2 nanocomposites have favorable biocompatibility and can reduced the ROS damage effectively. In vitro, the cck-8 analysis, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) stain and flow cytometry analysis showed rare negative influence by porous Se@SiO2 nanocomposites but significantly protective effect against H2O2 by reducing ROS level (detected by DCFH-DA). In vivo, the biosafety of porous Se@SiO2 nanocomposites were confirmed by the serum biochemistry, the ROS level in serum were significantly reduced and the curative effect were confirmed by Micro CT scan, serum Elisa assay (inflammatory factors), Western blotting (quantitative measurement of ONFH) and HE staining. It is expected that the porous Se@SiO2 nanocomposites may prevent steroid-induced ONFH by reducing oxidative stress. PMID:28256626

  17. Artemether Regulates Chemosensitivity to Doxorubicin via Regulation of B7-H3 in Human Neuroblastoma Cells.

    PubMed

    Tan, Wei-Qiang; Chen, Gang; Ye, Ming; Jia, Bing

    2017-09-03

    BACKGROUND Artemether, originally used for malaria, exhibits potential therapeutic efficacy against several types of cancer, including gastric cancer, hepatocellular carcinoma, and gliomas. In this study, we investigated the role and mechanism of artemether on drug resistance of neuroblastoma cells. MATERIAL AND METHODS Cell viability and proliferation were determined by CCK-8 and EdU incorporation assay, respectively. Gene expression was measured by real-time PCR and Western blot analysis. RESULTS Our results revealed that artemether treatment remarkably inhibited the proliferation of neuroblastoma cell lines SH-SY5Y, SK-N-SH, and SK-N-BE2. In addition, co-treatment of tumor cells with artemether and doxorubicin significantly reduced cell viability and DNA synthesis compared with doxorubicin-treated cells. On the molecular level, we found that combined treatment with artemether and doxorubicin suppressed the expression of B7-H3 both at the mRNA and protein levels. In addition, artemether failed to sensitize tumor cells to doxorubicin in SH-SY5Y cells overexpressing B7-H3. CONCLUSIONS Artemether-mediated inhibition of B7-H3 may contribute to doxorubicin sensitivity in neuroblastoma cells, suggesting that artemether could serve as a potential therapeutic option for neuroblastoma.

  18. Synthesis and biological activities of some human gastrin analogs.

    PubMed

    Lima-Leite, A C; Fulcrand, P; Galleyrand, J C; Berge, G; Aumelas, A; Bali, J P; Castel, J; Martinez, J

    1996-10-01

    The synthesis of analogs of the C-terminal tridecapeptide of gastrin is described. These pseudopeptide analogs were obtained either by replacing the C-terminal phenylalanine amide with 2-phenylethylalcohol or with 2-phenylethylamine, or by replacing the peptide bond between Trp and Leu, or between Leu and Asp with an aminomethylene (CH2NH). The ability of these compounds to stimulate gastric acid secretion in anesthetized rats and to inhibit binding of labeled CCK-8 to isolated cells from rabbit fundic mucosa was tested. [desPhe13, Leu11]-HG-12-I-beta-phenylethylester 33, [desPhe13, Leu11]-HG-12-II-beta-phenylethylester 38, [desPhe13, Leu11]-HG-12-I-beta-phenylethylamide 32, and [desPhe13, Leu11]-HG-12-II-beta-phenylethylamide 37 acted as gastrin receptor antagonists, while [Trp10-psi(CH2NH)-Leu11]-HG-13-I 31 and [Trp10-psi(CH2NH)-Leu11]-HG-13-II 36 acted as agonists. Unexpectedly, [Leu11-psi(CH2NH)-Asp12]-HG-13-I 30 and [Leu11-psi (CH2NH)-Asp12]-HG-13-II 35 were almost devoid of affinity for the gastrin receptor.

  19. A synthetic peptide derivative that is a cholecystokinin receptor antagonist.

    PubMed

    Lignon, M F; Galas, M C; Rodriguez, M; Laur, J; Aumelas, A; Martinez, J

    1987-05-25

    So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.

  20. ICAM-1-Targeted Liposomes Loaded with Liver X Receptor Agonists Suppress PDGF-Induced Proliferation of Vascular Smooth Muscle Cells

    NASA Astrophysics Data System (ADS)

    Huang, Xu; Xu, Meng-Qi; Zhang, Wei; Ma, Sai; Guo, Weisheng; Wang, Yabin; Zhang, Yan; Gou, Tiantian; Chen, Yundai; Liang, Xing-Jie; Cao, Feng

    2017-05-01

    The proliferation of vascular smooth muscle cells (VSMCs) is one of the key events during the progress of atherosclerosis. The activated liver X receptor (LXR) signalling pathway is demonstrated to inhibit platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation. Notably, following PDGF-BB stimulation, the expression of intercellular adhesion molecule-1 (ICAM-1) by VSMCs increases significantly. In this study, anti-ICAM-1 antibody-conjugated liposomes were fabricated for targeted delivery of a water-insoluble LXR agonist (T0901317) to inhibit VSMC proliferation. The liposomes were prepared by filming-rehydration method with uniform size distribution and considerable drug entrapment efficiency. The targeting effect of the anti-ICAM-T0901317 liposomes was evaluated by confocal laser scanning microscope (CLSM) and flow cytometry. Anti-ICAM-T0901317 liposomes showed significantly higher inhibition effect of VSMC proliferation than free T0901317 by CCk8 proliferation assays and BrdU staining. Western blot assay further confirmed that anti-ICAM-T0901317 liposomes inhibited retinoblastoma (Rb) phosphorylation and MCM6 expression. In conclusion, this study identified anti-ICAM-T0901317 liposomes as a promising nanotherapeutic approach to overcome VSMC proliferation during atherosclerosis progression.

  1. Cytotoxic Effects of One-step Self-etching Dental Adhesives on Human Periodontal Ligament Fibroblasts In Vitro.

    PubMed

    Sun, Fangfang; Mao, Peng; Wang, Cong; Shi, Chaowen; Nie, Rongrong; Han, Ningning; Han, Xiaodong

    2016-01-01

    To evaluate the potential cytotoxic effects of four one-step self-etching dental adhesives [Adper Easy One (AEO), iBond (IB), Clearfil S³ Bond (CSB), and G-Bond (GB)] on cultured human periodontal ligament fibroblasts. Cured adhesives were immersed in complete DMEM or deionized water and maintained at 37°C for 24 h, followed by sterilization. The deionized water-based extract was used for Fourier transform infrared spectroscopy analysis. The DMEM-based extract was diluted into various concentrations for cytotoxicity tests. The viability, integrity, and apoptosis of cultured human periodontal ligament fibroblasts upon treatment with the extracts were determined using the CCK-8 assay, microscopy, and flow cytometry. All of the four adhesives induced cell viability loss, cell morphology alteration, and cell death. GB showed the greatest cytotoxicity by inducing cell apoptosis and necrosis, while IB had the weakest cytotoxic effect on the cultured cells. All tested dental adhesives have significant adverse effects on cell viability. Therefore, precautions should be taken to protect the periodontal tissues when dental adhesives are applied in the clinic.

  2. [Induction of hepatic specification of human adipose-derived stem cells (hADSCs) in vitro].

    PubMed

    Wang, Min; Pei, Hai-yun; Guan, Li-dong; Nan, Xue; Bai, Ci-xian; Liu, Hui; Li, Bao-wei; Wang, Yun-fang; Pei, Xue-tao

    2009-07-01

    To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined. The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.

  3. Expression and clinical role of small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA) as a novel cell cycle protein in NSCLC.

    PubMed

    Xue, Qun; Lv, Liting; Wan, Chunhua; Chen, Buyou; Li, Mei; Ni, Tingting; Liu, Yifei; Liu, Yanhua; Cong, Xia; Zhou, Yiqun; Ni, Runzhou; Mao, Guoxin

    2013-09-01

    A small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) is a 35 kDa protein involved in a number of biological processes. However, the role of SGTA in non-small-cell lung cancer (NSCLC) tumorigenesis has never been elucidated. The purpose of this study was to determine whether SGTA could serve as a biomarker for stratification and prediction of prognosis in NSCLC. Small glutamine-rich tetratricopeptide repeat-containing protein alpha expression was evaluated by Western blot in 8 paired fresh lung cancer tissues and immunohistochemistry on 83 paraffin-embedded sections. The effect of SGTA was assessed by RNA interference in A549 cells. Serum starvation and refeeding, flow cytometry, CCK-8, and tunnel assays were performed. Small glutamine-rich tetratricopeptide repeat-containing protein alpha was highly expressed in NSCLC and significantly correlated with NSCLC histological differentiation, clinical stage, and Ki-67. Multivariate analysis indicated that SGTA was an independent prognostic factor for NSCLC patients' survival. The present investigation demonstrated that suppression of SGTA expression resulted in a significant decline of proliferation in A549 cells. Besides, SGTA could abolish the toxicity of cisplatin in A549 cells. These findings suggested that SGTA might play an important role in promoting the tumorigenesis of NSCLC, and thus be a promising therapeutic target to prevent NSCLC progression.

  4. Immunocytochemical and ultrastructural characterization of endocrine cells in the larval stomach of the frog Rana temporaria tadpoles: a comparison with adult specimens.

    PubMed

    Villaro, A C; Rovira, J; Bodegas, M E; Burrell, M A; García-Ros, D; Sesma, P

    2001-10-01

    According to immunostaining and ultrastructural patterns, Rana temporaria tadpole stomach displays a well-differentiated endocrine population comprising, at least, six cellular types: ECL, EC [serotonin], D [somatostatin] - all three of them abundant -, P [bombesin] - less numerous -, CCK-8 [cholecystokinin/gastrin] and A [glucagon/glicentin] - both very scarce. Larval endocrine cells are mainly located in the surface epithelium and show open or closed morphologies. Cellular diversity is similar in tadpoles and frogs, with the exception of immunoreactivity for gastrin-17, found in adults in numerous cells. Larval cells display mature ultrastructural traits, although with smaller secretory granules. The different distribution of endocrine cells, which in adults are preferentially located in the glands, probably refers to different functional requirements. However, the rich vascular plexus present in larval mucosa may be an efficient transport medium of surface hormones to-gastric targets. The enhancement in adults of endocrine population and correlative increase in hormonal secretion indicates a more active functional role, probably related to the shift from herbivorous to carnivorous habits. In summary, the tadpole gastric endocrine population, although not as numerous as that of adult frogs, displays histological traits that indicate a relevant (immunoreactive and ultrastructural properties, cellular diversity) and specific (surface location, relative abundance of open-type cells) role of local regulatory factors in amphibian larval gastric function.

  5. In vitro and in vivo evaluations of nano-hydroxyapatite/polyamide 66/glass fibre (n-HA/PA66/GF) as a novel bioactive bone screw.

    PubMed

    Su, Bao; Peng, Xiaohua; Jiang, Dianming; Wu, Jun; Qiao, Bo; Li, Weichao; Qi, Xiaotong

    2013-01-01

    In this study, we prepared nano-hydroxyapatite/polyamide 66/glass fibre (n-HA/PA66/GF) bioactive bone screws. The microstructure, morphology and coating of the screws were characterised, and the adhesion, proliferation and viability of MC3T3-E1 cells on n-HA/PA66/GF scaffolds were determined using scanning electron microscope, CCK-8 assays and cellular immunofluorescence analysis. The results confirmed that n-HA/PA66/GF scaffolds were biocompatible and had no negative effect on MC3T3-E1 cells in vitro. To investigate the in vivo biocompatibility, internal fixation properties and osteogenesis of the bioactive screws, both n-HA/PA66/GF screws and metallic screws were used to repair intercondylar femur fractures in dogs. General photography, CT examination, micro-CT examination, histological staining and biomechanical assays were performed at 4, 8, 12 and 24 weeks after operation. The n-HA/PA66/GF screws exhibited good biocompatibility, high mechanical strength and extensive osteogenesis in the host bone. Moreover, 24 weeks after implantation, the maximum push-out load of the bioactive screws was greater than that of the metallic screws. As shown by their good cytocompatibility, excellent biomechanical strength and fast formation and ingrowth of new bone, n-HA/PA66/GF screws are thus suitable for orthopaedic clinical applications.

  6. Effects of S100A6 gene silencing on the biological features of eutopic endometrial stromal cells and β-catenin expression

    PubMed Central

    Zhang, Xiaoling; Liu, Zequn; Chen, Meihong; Cao, Qing; Huang, Donghua

    2017-01-01

    Protein expression levels of S100 calcium binding protein A6 (S100A6) are increased in various malignancies and are associated with tumor behavior; however, the association between S100A6 and endometriosis remains to be elucidated. In order to investigate the influence of S100A6 protein, recombinant lentivirus siS100A6 was used to transfect the eutopic endometrial stromal cells. CCK-8 assay was performed to identify the proliferation ability of cell and the cell migration was detected by Transwell assay. Flow cytometry was performed to detect cell apoptosis, and western blotting and reverse transcription-quantitative polymerase chain reaction were performed to identify the expression of β-catenin. The present study investigated the role of S100A6 in endometriosis and its interaction with β-catenin by transfecting eutopic endometrial stromal cells with a recombinant lentivirus containing S100A6-specific small interfering RNA. Inhibition of S100A6 expression had a significant antiproliferative effect and reduced the migratory ability of eutopic endometrial stromal cells, and induced their apoptosis. In addition, inhibition of S100A6 expression suppressed β-catenin expression. These results suggested that inhibition of S100A6 may represent a promising novel approach for the targeted therapy of endometriosis. PMID:28075439

  7. Enhanced bioreduction-responsive biodegradable diselenide-containing poly(ester urethane) nanocarriers.

    PubMed

    Wei, Chao; Zhang, Yan; Song, Zhongchen; Xia, Yiru; Xu, Heng; Lang, Meidong

    2017-02-03

    Stimuli-responsive nanocarriers have been limited for bench-to-bedside translation mainly because the stimuli sensitivity and responsive rate are not high enough to ensure sufficient drug concentration at the target sites for superior therapeutic benefits. Herein, we reported an enhanced bioreduction-responsive and biodegradable nanocarrier based on the amphiphilic poly(ester urethane) copolymers (PAUR-SeSe) bearing multiple diselenide groups on the backbone. The copolymer could spontaneously self-assemble into stable micelles in aqueous medium with an average diameter of 68 nm, which could be rapidly disassembled in a reductive environment as a result of the reduction-triggered cleavage of diselenide groups. Furthermore, the PAUR-SeSe micelles showed an enhanced drug release profile and cellular uptake compared with the disulfide-containing analogue (PAUR-SS). CCK8 assays revealed that the antitumor activity of DOX-loaded PAUR-SeSe micelles was much higher than that of DOX-loaded PAUR-SS micelles. Besides, the blank micelles and degradation products were nontoxic up to a tested concentration of 50 μg mL(-1). Therefore, the enhanced therapeutic efficacy and good biocompatibility demonstrated that this drug nanocarrier had great potential for smart antitumor drug delivery applications.

  8. Low-power laser irradiation inhibits amyloid beta-induced cell apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Heng; Wu, Shengnan

    2011-03-01

    The deposition and accumulation of amyloid-β-peptide (Aβ) in the brain are considered a pathological hallmark of Alzheimer's disease(AD). Apoptosis is a contributing pathophysiological mechanism of AD. Low-power laser irradiation (LPLI), a non-damage physical therapy, which has been used clinically for decades of years, is shown to promote cell proliferation and prevent apoptosis. Recently, low-power laser irradiation (LPLI) has been applied to moderate AD. In this study, Rat pheochromocytoma (PC12) cells were treated with amyloid beta 25-35 (Aβ25-35) for induction of apoptosis before LPLI treatment. We measured cell viability with CCK-8 according to the manufacture's protocol, the cell viability assays show that low fluence of LPLI (2 J/cm2 ) could inhibit the cells apoptosis. Then using statistical analysis of proportion of apoptotic cells by flow cytometry based on Annexin V-FITC/PI, the assays also reveal that low fluence of LPLI (2 J/cm2 ) could inhibit the Aβ-induced cell apoptosis. Taken together, we demonstrated that low fluence of LPLI (2 J/cm2 ) could inhibit the Aβ-induced cell apoptosis, these results directly point to a therapeutic strategy for the treatment of AD through LPLI.

  9. Thermoresponsive Polymers with Lower Critical Solution Temperature- or Upper Critical Solution Temperature-Type Phase Behaviour Do Not Induce Toxicity to Human Endothelial Cells.

    PubMed

    Ji, Yuejia; Zhu, Mengxiang; Gong, Yu; Tang, Haoyu; Li, Juan; Cao, Yi

    2017-01-01

    Thermoresponsive polymers have gained extensive attention as biomedical materials especially for targeted drug delivery systems. We have recently developed water-soluble polypeptide-based thermoresponsive polymers that exhibit lower critical solution temperature (LCST)- or upper critical solution temperature (UCST)-type phase behaviours. In this study, the toxicity of these polymers to human umbilical vein endothelial cells (HUVECs) was investigated to assess the safety and biocompatibility. Up to 100 μg/ml, thermoresponsive polymers did not induce cytotoxicity to HUVECs, showing as unaltered mitochondrial viability assessed as cell counting kit-8 (CCK-8) assay and membrane integrity assessed as lactate dehydrogenase (LDH) assay. Inflammatory response, assessed as the release of chemokine-soluble monocyte chemotactic protein 1 (sMCP-1) and interleukin-8 (IL-8) as well as cytokine IL-6, was not significantly affected by the polymers. In addition, 1 μM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, significantly decreased mitochondrial viability, but did not affect membrane integrity or inflammatory response. The presence of thermoresponsive polymers with LCST-type phase behaviour did not further affect the effects of TG. In conclusion, the thermoresponsive polymers used in this study are not toxic to endothelial cells and therefore could be further considered as safe materials for biomedical applications.

  10. [Effect of lycium bararum polysaccharides on angiotensin II-induced senescence of human umbilical vein endothelial cells and expressions of P53 and P16].

    PubMed

    Liu, Ling; Wang, Xue-ni; Liu, Ze; Wang, Lu-ni; Wu, Jun; Wang, Wei; Feng, Ju-xiang

    2011-06-01

    To investigate the role of lycium bararum polysaccharides (LBP) on angiotensin II (AngII)-induced senescence of human umbilical vein endothelial cells (HUVECs) and expressions of P53 and P16 and explore the mechanism of LBP against aging. HUVECs cultured in vitro were stimulated with 1×10(-6) mmol/L AngII to induce cell senescence, which was identified using β-gal staining. Flow cytometry was used for analyzing the cell cycle changes, and the cell viability was assessed using CCK-8 method. Western blotting was employed to detect the expression of P53 and P16 in the exposed cells. Compared with the control cells, the cells positive for β-gal staining was significantly increased in AngII group, and showed cell cycle arrest at G(0)/G(1) phase with decreased S-phase cell percentage and cell viability. The expression levels of P53 and P16 were significantly increased in the cells with AngII exposure (P<0.05). LBP treatment of AngII-exposed cells resulted in decreased β-gal-positive cells with a reduction in G(0)/G(1) phase cells and an increase in S phase cells. LBP treatment also increased the cell viability and significantly decreased the expression levels of P53 and P16 (P<0.05). LBP can delay AngII-induced aging of HUVECs possibly by down-regulating the expression of P53 and P15.

  11. The effects of PK11195 on meningioma was associated with allopregnanolone biosynthesis, which was mediated by translocator protein 18 KDa.

    PubMed

    Gao, Zhuo-Wei; Huang, Jing-Bin; Lin, Qing; Qin, Qiang; Liang, Yao-Jun; Zhou, Lu; Luo, Min

    2016-01-01

    Meningioma is one of the common brain tumors in adults. It had been shown that the allopregnanolone biosynthesis was associated with tumorigenesis and PK11195, the translocator protein 18 KDa (TSPO) antagonist, had the effects of the allopregnanolone biosynthesis. However, little is known about the association between the effects of PK11195 on meningioma and the allopregnanolone biosynthesis. To evaluate this, the meningioma cell line IOMM-LEE was applied. Cell viability and proliferation were determined by CCK-8 assay. The IC50 of PK11195 on the IOMM-LEE was 1.505 ± 0.08 nM. The cell viability and proliferation of AC-5216 (TSPO selective ligand, 2 and 4 nM) was blocked by PK11195 (1.5 nM). Further, we evaluated the role of allopregnanolone biosynthesis in the effects of TSPO on meningioma. Enzyme-Linked ImmunoSorbent Assay (ELISA) was used in the measurement of the allopregnanolone level. It showed that the allopregnanolone level was increased by AC-5216 (2 and 4 nM) and the increase was reversed by PK11195 (1.5 nM). Collectedly, it firstly indicated that the effects of PK11195 on meningioma were relevant to the decrease of allopregnanolone biosynthesis, which was mediated by TSPO.

  12. Photoresponsive lipid-polymer hybrid nanoparticles for controlled doxorubicin release

    NASA Astrophysics Data System (ADS)

    Yao, Cuiping; Wu, Ming; Zhang, Cecheng; Lin, Xinyi; Wei, Zuwu; Zheng, Youshi; Zhang, Da; Zhang, Zhenxi; Liu, Xiaolong

    2017-06-01

    Currently, photoresponsive nanomaterials are particularly attractive due to their spatial and temporal controlled drug release abilities. In this work, we report a photoresponsive lipid-polymer hybrid nanoparticle for remote controlled delivery of anticancer drugs. This hybrid nanoparticle comprises three distinct functional components: (i) a poly(D,L-lactide-co-glycolide) (PLGA) core to encapsulate doxorubicin; (ii) a soybean lecithin monolayer at the interface of the core and shell to act as a molecular fence to prevent drug leakage; (iii) a photoresponsive polymeric shell with anti-biofouling properties to enhance nanoparticle stability, which could be detached from the nanoparticle to trigger the drug release via a decrease in the nanoparticle’s stability under light irradiation. In vitro results revealed that this core-shell nanoparticle had excellent light-controlled drug release behavior (76% release with light irradiation versus 10% release without light irradiation). The confocal microscopy and flow cytometry results also further demonstrated the light-controlled drug release behavior inside the cancer cells. Furthermore, a CCK8 assay demonstrated that light irradiation could significantly improve the efficiency of killing cancer cells. Meanwhile, whole-animal fluorescence imaging of a tumor-bearing mouse also confirmed that light irradiation could trigger drug release in vivo. Taken together, our data suggested that a hybrid nanoparticle could be a novel light controlled drug delivery system for cancer therapy.

  13. Macrophage migration inhibitory factor enhances lipopolysaccharide-induced fibroblast proliferation by inducing toll-like receptor 4.

    PubMed

    Xi, Zheng-de; Xie, Chang-yi; Xi, Ye-bin

    2016-01-26

    Fibroblast proliferation is a common manifestation of chronic inflammatory diseases, including rheumatoid arthritis (RA), Crohn's disease and ulcerative colitis, etc. To alleviate patient suffering, the mechanism underlying fibroblast proliferation should be elucidated. CCK-8 assay was used to assess the stimulatory effect of LPS and macrophage migration inhibitory factor (MIF) on fibroblast proliferation. Then, TLR4 expression on fibroblast cell membrane was carried out by confocal scanning microscopy. Finally, real-time fluorescent quantitative PCR and flow cytometry were applied to determine the expression of TLR4 after MIF challenge. LPS alone directly stimulated the fibroblast proliferation. In addition, MIF showed co-stimulatory effect on LPS-induced fibroblast proliferation. Interestingly, fibroblast overtly expressed TLR4 without stimulation. After MIF stimulation, real-time PCR showed TLR4 mRNA levels were increased by about 33% in the fibroblasts; in agreement, TLR4 expression on the fibroblast membrane was increased by about 20%, as shown by flow cytometry. These findings indicated MIF elevates TLR4 expression in fibroblast, enhancing LPS-induced cell proliferation.

  14. Mechanistic Study of the Inhibitory Effect of Kaempferol on Uterine Fibroids In Vitro.

    PubMed

    Li, Yanxia; Ding, Zhaoxia; Wu, Chuanzhong

    2016-12-08

    BACKGROUND This study examined the effect of kaempferol on uterine fibroids in vitro and the underlying mechanism, and investigated the potential of kaempferol as a clinical drug for the treatment of uterine fibroids. MATERIAL AND METHODS Uterine fibroid tissue and surrounding smooth muscle tissue were collected for primary culture. Different concentrations of kaempferol (12 μM, 24 μM, and 48 μM) were used to treat the cells for 24, 48, and 72 hours. Ethanol was used in the control group. A CCK-8 colorimetric assay was used to detect cell proliferation. Real-time PCR and immunoblot were used to detect estrogen receptor (ER), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF) levels in mRNA and protein. RESULTS The differences in proliferation at different time points and concentrations of kaempferol were statistically significant. The inhibitory effect of kaempferol on mRNA levels of ER and IGF, and protein levels of ER, VEGF, and IGF-1 were positively correlated with kaempferol concentration. Changes in kaempferol concentration showed no effect on VEGF mRNA expression. Treatment with kaempferol significantly lowered myocardin levels in uterine fibroid tissue compared to normal uterine smooth muscle (P<0.05). CONCLUSIONS Kaempferol might be used for clinical treatment of uterine fibroids due to its inhibitory effect on the proliferation of uterine fibroids cells.

  15. Kaempferol inhibited VEGF and PGF expression and in vitro angiogenesis of HRECs under diabetic-like environment.

    PubMed

    Xu, X H; Zhao, C; Peng, Q; Xie, P; Liu, Q H

    2017-03-02

    Diabetic retinopathy (DR) is one of the common and specific microvascular complications of diabetes. This study aimed to investigate the anti-angiogenic effect of kaempferol and explore its underlying molecular mechanisms. The mRNA expression level of vascular endothelial growth factor (VEGF) and placenta growth factor (PGF) and the concentrations of secreted VEGF and PGF were measured by qTR-PCR and ELISA assay, respectively. Human retinal endothelial cells (HRECs) proliferation, migration, and sprouting were measured by CCK-8 and transwell, scratching wound, and tube formation assays, respectively. Protein levels were determined by western blot. High glucose (25 mM) increased the mRNA expression levels of VEGF and PGF as well as the concentrations of secreted VEGF and PGF in HRECs, which can be antagonized by kaempferol (25 µM). Kaempferol (5-25 µM) significantly suppressed cell proliferation, migration, migration distance and sprouting of HRECs under high glucose condition. The anti-angiogenic effect of kaempferol was mediated via downregulating the expression of PI3K and inhibiting the activation of Erk1/2, Src, and Akt1. This study indicates that kaempferol suppressed angiogenesis of HRECs via targeting VEGF and PGF to inhibit the activation of Src-Akt1-Erk1/2 signaling pathway. The results suggest that kaempferol may be a potential drug for better management of DR.

  16. Cisplatin induces expression of drug resistance-related genes through c-jun N-terminal kinase pathway in human lung cancer cells.

    PubMed

    Xu, Li; Fu, Yingya; Li, Youlun; Han, Xiaoli

    2017-08-01

    Change of multidrug resistance-related genes (e.g., lung resistance protein, LRP) and overexpression of anti-apoptotic genes (Bcl-2, Bcl-Xl, XIAP, Survivin) are responsible for cisplatin resistance. In our study, we investigated the mechanism by which cisplatin induces LRP, Bcl-2, Bcl-xL, XIAP, and Survivin expression in human lung adenocarcinoma A549 cells and human H446 small cell lung cancer cells at mRNA and protein levels. In our study, cell proliferation was assessed with CCK-8 assays, and cell apoptosis was assessed with flow cytometric analysis and Annexin-V/PI staining. qPCR was used to complete RNA experiments. Protein expression was assessed with Western blotting. Cisplatin increased Bcl-2, LRP, and Survivin expression, but decreased Bcl-xL and XIAP expression in a dose-dependent manner. Preincubation with JNK-specific inhibitor, SP600125, significantly inhibited these genes' expression at mRNA and protein levels, enhanced chemosensitivity of lung cancer cells to cisplatin, and promoted cisplatin-induced apoptosis. Our data suggest that the JNK signaling pathway plays an important role in cisplatin resistance. Lung resistance protein (LRP) and anti-apoptotic genes (Bcl-2, Bcl-Xl, XIAP, Survivin) are involved in the process. The results reminded us of a novel therapy target for lung cancer treatment.

  17. Expression patterns of three regulation enzymes in glycolysis in esophageal squamous cell carcinoma: association with survival.

    PubMed

    Li, Wenfeng; Xu, Zhengyang; Hong, Junfeng; Xu, Yunsheng

    2014-09-01

    Enhanced glycolysis is a common trait of many types of human cancers. This study was to detect the expression pattern of three regulatory enzymes during glycolysis in esophageal squamous cell carcinoma (ESCC) and to investigate their correlation with patients' outcome based on banked pathology material. A total of 141 surgically resected specimens of primary ESCC patients without prior treatments were retrospectively recruited from the First Affiliated Hospital of Wenzhou Medical College Hospital from 2007 to 2009. Expression of HK1, PFKB, and PKM2 in ESCC specimens was analyzed by immunohistochemical staining and Western blotting analysis. HK1-shRNA was used to knock down HK1 expression in ESCC cells, and the functional significance was assessed by CCK8 assay. It was found that the expression of two glycolytic enzymes, HK1 and PKM2, was associated with disease progression, invasion, and poor survival of patients with ESCC. Silence of HK1-inhibited cell proliferation in vitro and suppressed phospho-S6 kinase expression. Our findings suggest that activation of key enzymes in glycolysis might serve as potential therapeutic targets and/or prognostic factors for patients with ESCC.

  18. Long noncoding RNA linc00346 promotes the malignant phenotypes of bladder cancer.

    PubMed

    Ye, Tingyu; Ding, Wei; Wang, Nanxiong; Huang, Hang; Pan, Yue; Wei, Anyang

    2017-09-09

    More and more reports have demonstrated that long noncoding RNAs (lncRNAs) play an important role in the development of a variety of carcinomas, including bladder cancer. However, only a small fraction of them have been characterized. Linc00346 have been found to be upregulated in bladder cancer tissues compared to normal tissues in a microarray-based lncRNA profiling study. In this study, we would like to explore the expression pattern and functional role of linc00346 in bladder cancer. We determined the expression of linc00346 in a cohort of bladder cancer tissues with matched normal tissues as well as human bladder cancer cell lines. We investigated the biological function of linc00346 with CCK-8 assay, colony formation assay, flow cytometry analysis, transwell assay and tumor xenografts mice model. We found that linc00346 was upregulated in bladder cancer tissues compared to normal tissues. Knockdown of linc00346 inhibited bladder cancer cell proliferation and migration, induced cell cycle arrest and cell apoptosis. Our study demonstrates that linc00346 could be a potential oncogene and a therapeutic target in bladder cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Nicotine may promote tongue squamous cell carcinoma progression by activating the Wnt/β-catenin and Wnt/PCP signaling pathways.

    PubMed

    Wang, Chengze; Xu, Xin; Jin, Hairu; Liu, Gangli

    2017-05-01

    To investigate the effects and the possible underlying mechanisms of nicotine stimulation on tongue squamous cell carcinoma (TSCC) progression, a TSCC cell line Cal27 and 34 samples of paraffin-embedded TSCC were examined. Immunofluorescence, western blot analysis, and TOP/FOP flash, CCK-8, wound healing and Transwell invasion assays were used to evaluate Cal27 in response to nicotine stimulation. We also investigated expression levels of related proteins of Wnt/β-catenin and Wnt/PCP pathways in paraffin-embedded TSCC samples with or without a history of smoking by immunohistochemistry. Nicotine stimulation can promote proliferation, migration, and invasion of TSCC cells in vitro, downregulate E-cadherin, and activate the Wnt/β-catenin and Wnt/PCP pathways, which could be antagonized by the α7 nicotine acetylcholine receptor (α7 nAChR) inhibitor α-BTX. Moreover, the expression levels of β-catenin, Wnt5a and Ror2 were higher in TSCC patients with a history of smoking than those without a history of smoking. Our results suggest nicotine may promote tongue squamous carcinoma cells progression by activating the Wnt/β-catenin and Wnt/PCP signaling pathways and may play a significant role in the progression and metastasis of smoking-related TSCC.

  20. Enhancement of radiosensitivity in human esophageal carcinoma cells by fenofibrate and its potential mechanism.

    PubMed

    Li, Xiao-Qing; Zhou, Jun-Dong; Zou, Shi-Tao; Yu, Jian; Meng, Xin-Jun; Wu, Jin-Chang

    2015-01-01

    Fenofibrate is a specific agonist of PPARα, and is characterized by relatively low systemic toxicity. Recent studies have revealed that fenofibrate suppresses the growth of several cancer lines in vitro, but the exact relation between fenofibrate and irradiation has not been explored. The purpose of this study was to investigate the radiosensitivity enhancement effects of fenofibrate combined with radiation on the human esophageal carcinoma cell lines Eca-109 and TE1, and the potential mechanism underlying these effects. The Eca-109 and TE1 cell lines were tested by the CCK-8 assay for cell proliferation. The multitarget click model was used to delineate the survival curve and radiosensitivity was determined after cells were treated with fenofibrate and/or x-ray radiation. Flow cytometry was used to examine the effect of fenofibrate and radiation on the cell cycle. The expression of vascular endothelial growth factor (VEGF) protein was detected by Western blot analysis. When given alone, fenofibrate had a time- and concentration-dependent cytotoxic effect on cells. The dose-enhancement ratio for combined fenofibrate and radiation increased markedly compared with fenofibrate alone. Further, the ratio of cells in the G2/M phase after fenofibrate and radiation was higher than that after fenofibrate or irradiation alone. The expression of VEGF protein was suppressed after treatment with fenofibrate alone or fenofibrate plus radiation. Fenofibrate can enhance the ra