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Sample records for bromodeoxyuridine

  1. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J;

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogona...

  2. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J;

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal...... light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could...... fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses....

  3. Intra-arterial bromodeoxyuridine radiosensitization of malignant gliomas

    International Nuclear Information System (INIS)

    In the 1950's it was first observed that mammalian cells exposed to the halogenated deoxyuridines were more sensitive to ultraviolet light and radiation than untreated cells. This prompted early clinical trials with bromodeoxyuridine (BUdR) which showed mixed results. More recently, several Phase I studies, while establishing the feasibility of continuous intravenous (IV) infusion of BUdR, have reported significant dose limiting skin and bone marrow toxicities and have questioned the optimal method of BUdR delivery. To exploit the high mitotic activity of malignant gliomas relative to surrounding normal brain tissue, we have developed a permanently implantable infusion pump system for safe, continuous intraarterial (IA) internal carotid BUdR delivery. Since July 1985, 23 patients with malignant brain tumors (18 grade 4, 5 grade 3) have been treated in a Phase I clinical trial using IA BUdR (400-600 mg/m2/day X 8 1/2 weeks) and focal external beam radiotherapy (59.4 Gy at 1.8 Gy/day in 6 1/2 weeks). Following initial biopsy/surgery the infusion pump system was implanted; BUdR infusion began 2 weeks prior to and continued throughout the 6 1/2 week course of radiotherapy. There have been no vascular complications. Side-effects in all patients have included varying degrees of anorexia, fatigue, ipsilateral forehead dermatitis, blepharitis, and conjunctivitis. Myelosuppression requiring dose reduction occurred in one patient. An overall Kaplan-Meier estimated median survival of 20 months has been achieved. As in larger controlled series, histologic grade and age are prognostically significant. We have shown in a Phase I study that IA BUdR radiosensitization is safe, tolerable, may lead to improved survival, and appears to be an efficacious primary treatment of malignant gliomas

  4. Continuous intravenous infusions of bromodeoxyuridine as a clinical radiosensitizer

    International Nuclear Information System (INIS)

    Twelve patients were treated with continuous intravenous (24-hour) infusions of bromodeoxyuridine (BUdR) at 650 or 1000 mg/m2/d for up to two weeks. Myelosuppression, especially thrombocytopenia, was the major systemic toxicity and limited the infusion period to nine to 14 days. However, bone marrow recovery occurred within seven to ten days, allowing for a second infusion in most patients. Local toxicity (within the radiation field) was minimal, with the exception of one of four patients, who underwent abdominal irradiation. Pharmacology studies revealed a steady-state arterial plasma level of 6 x 10(-7) mol/L and 1 x 10(-6) mol/L during infusion of 650 and 1000 mg/m2/d, respectively. In vivo BUdR uptake into normal bone marrow was evaluated in two patients by comparison of preinfusion and postinfusion in vitro radiation survival curves of marrow CFUc with enhancement ratios (D0-pre/D0-post) of 1.8 (with 650 mg/m2/d) and 2.5 (with 1000 mg/m2/d). In vivo BUdR incorporation into normal skin and tumor cells using an anti-BUdR monoclonal antibody and immunohistochemistry was demonstrated in biopsies from three patients revealing substantially less cellular incorporation into normal skin (less than 10%) compared with tumor (up to 50% to 70%). The authors conclude that local and systemic toxicity of continuous infusion of BUdR at 1000 mg/m2/d for approximately two weeks is tolerable. The observed normal tissue toxicity is comparable with previous clinical experience with intermittent (12 hours every day for two weeks) infusions of BUdR. Theoretically, a constant infusion should allow for greater incorporation of BUdR into cycling tumor cells and thus, for further enhancement of radiosensitization

  5. EXAMINATION OF THE MICROBIAL ECOLOGY OF THE AVIAN INTESTINE IN VIVO USING BROMODEOXYURIDINE

    Science.gov (United States)

    Bromodeoxyuridine (BrdU), a thymidine analog that can be incorporated into the DNA of actively dividing cells, has been used in vivo to identify bacteria that are metabolically active during an acute period of feed withdrawal in three-week old turkey poults. The microbiota in the ceca were determine...

  6. Bromodeoxyuridine combined with UV light and gamma irradiation promotes the production of asymmetric somatic hybrid calli

    International Nuclear Information System (INIS)

    The degree of gamma‐ or X‐ray‐induced donor chromosome elimination in asymmetric somatic hybrids is highly variable. Here the beneficial use of bromodeoxyuridine and UV light as additional chromosome destabilizing agents is described. Protoplasts of Nicotiana tabacum were fused with protoplasts of Nicotiana plumbaginifolia (Np) that carried the kanamycin‐resistance and glucuronidase (GUS) genes on separate chromosomes. Prior to fusion, the Np donor protoplasts were pretreated with bromodeoxyuridine and then were inactivated by treatment with iodoacetate ± UV light ± 200 Gy gamma irradiation. Hybrids were selected on medium containing kanamycin. The elimination of Np DNA was assessed by scoring of the fraction of hybrid calli that expressed GUS and by dot‐blot analysis using a Np‐specific probe. gamma irradiation alone resulted in elimination of 50% of Np DNA. Pretreatment with bromodeoxyuridine (10 μM) followed by 2.5 to 5 min UV light resulted in the elimination of 35–45% of the donor genome, but incorporation of bromodeoxyuridine (10 μM) followed by 2,5 to 5 min UV light and 200 Gy gamma irradiation resulted in 85 to 90% elimination of Np DNA

  7. Cell proliferation in the developing rat pineal gland.A bromodeoxyuridine immunohistochemical study

    OpenAIRE

    Calvo, J.L.; Boya, J; Carbonell, A L; García-Mauriño, J.E.

    2000-01-01

    The immunohistochemical detection of bromodeoxyuridine (BrdU) was used to study the cell proliferation in the developing rat pineal gland, from the appearance of pineal primordium in the embryonic day 15 (E15) until 30 days after birth. The results showed three different proliferative phases. From E15 to E21, the pineal gland shows a phase of rapid proliferation. The second phase corresponds to the first postnatal week, in which the number of labeled cells per ...

  8. Role of bromodeoxyuridine in the formation of SCE after γ-irradiation of human lymphocytes at the presynthetic stage of the cell cycle

    International Nuclear Information System (INIS)

    The formation of SCE was studied in human lymphocytes irradiated at the presynthetic stage of the cell cycle in the presence or absence of 5-bromodeoxyuridine. The results obtained showed that 5-bromodeoxyuridine present in the cultur medium at the time of exposure or during the postirradiation period increased the yield of SCE. It is suggested that 5-bromodeoxyuridine exerts its effect through interfering with DNA repair

  9. Replicon sizes in non-transformed and SV40-transformed cells, as estimated by a bromodeoxyuridine photolysis method

    International Nuclear Information System (INIS)

    Replicon sizes were measured in Simian Virus 40 (SV40)-transformed and untransformed normal human, xeroderma pigmentosum (XP), and mouse 3T3 cells with an x-ray plus bromodeoxyuridine (BUdR) photolysis method. Replicon sizes in SV40-transformed cells were at least twice those in untransformed counterparts, but DNA fork displacement rates were only slightly increased

  10. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    Energy Technology Data Exchange (ETDEWEB)

    Muir, D.; Varon, S.; Manthorpe, M. (Univ. of California, San Diego, La Jolla (USA))

    1990-03-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

  11. Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J; Christiansen, J;

    1993-01-01

    Nuclear RNA synthesis can be analysed by flow cytometry of cells labelled with 5-bromouridine (BrUrd) and stained with anti-bromodeoxyuridine (BrdUrd) antibody and FITC-conjugated secondary antibody. A panel of 5 different commercially available anti-BrdUrd antibodies was tested on cells of a HL-60...... human leukemia cell line, stained as a methanol-fixed nuclear suspension. The BrUrd-induced fluorescence signals were highest with the antibody ABDM (Partec), moderate but reproducible with B-44 (Becton Dickinson), variable or low with BR-3 and IU-4 (Caltag), and not detectable with Bu20a (DAKO...... variation of RNA synthesis during the cell cycle. The BrUrd incorporation was high in the S and G2 phase, variable in G1, and negligible in mitosis. Similar results were obtained using other cell types....

  12. Bromodeoxyuridine-Labeled Viral Particles as a Tool for Visualization of the Immediate-Early Events of Human Cytomegalovirus Infection

    OpenAIRE

    Rosenke, Kyle; Fortunato, Elizabeth A.

    2004-01-01

    We describe here a simple method for labeling the genome of human cytomegalovirus, a large double-stranded DNA virus, with bromodeoxyuridine (BrdU). The labeled DNA was incorporated into viral particles, which were then collected in cell supernatant. To demonstrate the versatility and effectiveness of this method, labeled virions were used to study the immediate-early events of virus-host cell interaction via indirect immunofluorescence microscopy. It is our hope that this new methodology wil...

  13. Replicon sizes in mammalian cells as estimated by an x-ray plus bromodeoxyuridine photolysis method

    International Nuclear Information System (INIS)

    A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [3H]thymidine ([3H]TdR) and bromodeoxyuridine (BrDUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, L/sub obs/, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of x rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions L/sub obs/ continues to increase only until operating replicons have completed their replication. This value for L/sub obs/ then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before x irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 μM, whereas for Chinese hamster ovary cells, the average replicon size is about 42 μM

  14. Differences in the incorporation of bromodeoxyuridine by human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, E.E.; Strauss, B.

    1975-08-01

    Long term human lymphoblastoid lines differ in their ability to grow in medium containing bromodeoxyuridine (BrdU) and to incorporate analog into their DNA. Eight Burkitts' lymphoma cell lines divided at least twice in BrdU-containing medium and made DNA in which over 90 percent of the thymidine residues were substituted with analog in both strands. Three infectious mononucleosis-derived lines and 24 lines transformed in vitro were inhibited by BrdU after one cell division and made only hybrid DNA in which one strand was substituted with analog. One out of eight normal individuals from whom long term lines were prepared gave cell lines which divided at least twice in BrdU and gave DNA in which both strands were substituted with analog. It would appear that intrinsic cellular factors regulate the response to BrdU and that Burkitt's tumor lines are characterized by their ability to make stable doubly substituted DNA containing a high proportion of halogenated analog.

  15. Identification of active oxalotrophic bacteria by Bromodeoxyuridine DNA labeling in a microcosm soil experiments.

    Science.gov (United States)

    Bravo, Daniel; Martin, Gaëtan; David, Maude M; Cailleau, Guillaume; Verrecchia, Eric; Junier, Pilar

    2013-11-01

    The oxalate-carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12 days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences), Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils. PMID:24033776

  16. Bromodeoxyuridine labeling index in glioblastoma multiforme: relation to radiation response, age, and survival

    International Nuclear Information System (INIS)

    Purpose: Various measures of the rate of tumor cell proliferation have been found to predict survival in patients with intracerebral gliomas. We correlated the bromodeoxyuridine labeling index (BrdUrd LI) with the response to radiation therapy, survival, and known prognostic factors in a series of patients with glioblastoma multiforme (GM) to test its utility as a prognostic factor. Methods and Materials: The BrdUrd LI was determined in 200 newly diagnosed intracranial GMs. Age and sex were known for all patients. The response to radiation therapy was determined in 116 patients by comparing neuroimaging studies obtained before and after external beam radiation therapy. Survival was analyzed in 64 patients who were treated according to two consecutive prospective clinical protocols. Results: The median BrdUrd LI was 6.5% (mean, 7.2%; range, 1.1-25.4%). The BrdUrd LI did not correlate significantly with age, sex, radiation response, or survival. Age and Karnofsky performance score were independent prognostic factors in our cohort. Conclusion: The proliferative rate as measured by BrdUrd LI was not a prognostic factor in our GM cohort. The BrdUrd LI did not correlate significantly with known prognostic factors in GM. There was no significant relationship between the BrdUrd LI and radiation response

  17. Use of bromodeoxyuridine immunocapture to identify psychrotolerant phenanthrene-degrading bacteria in phenanthrene-enriched polluted Baltic Sea sediments

    Energy Technology Data Exchange (ETDEWEB)

    Edlund, A.; Jansson, J.

    2008-05-01

    The aim of this study was to enrich and identify psychrotolerant phenanthrenedegrading bacteria from polluted Baltic Sea sediments. Polyaromatic hydrocarbon (PAH)-contaminated sediments were spiked with phenanthrene and incubated for 2 months in the presence of bromodeoxyuridine that is incorporated into the DNA of replicating cells. The bromodeoxyuridine-incorporated DNA was extracted by immunocapture and analyzed by terminal-restriction fragment length polymorphism and 16S rRNA gene cloning and sequencing to identify bacterial populations that were growing. In addition, degradation genes were quantified in the bromodeoxyuridine-incorporated DNA by real-time PCR. Phenanthrene concentrations decreased after 2 months of incubation in the phenanthrene-enriched sediments and this reduction correlated to increases in copy numbers of xylE and phnAc dioxygenase genes. Representatives of Exiguobacterium, Schewanella,Methylomonas, Pseudomonas, Bacteroides and an uncultured Deltaproteobacterium and a Gammaproteobacterium dominated the growing community in the phenanthrene spiked sediments. Isolates that were closely related to three of these bacteria (two pseudomonads and an Exiguobacterium sp.) could reduce phenanthrene concentrations in pure cultures and they all harbored phnAc dioxygenase genes. These results confirm that this combination of culture-based and molecular approaches was useful for identification of actively growing bacterial species with a high potential for phenanthrene degradation.

  18. Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

    International Nuclear Information System (INIS)

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis

  19. A phase I trial of intravenous bromodeoxyuridine and radiation therapy for pancreatic cancer

    International Nuclear Information System (INIS)

    Purpose: Improved radiosensitization may lead to improved results of treatment for pancreatic cancer. This Phase I trial was designed to determine the maximum tolerable dose of intravenous bromodeoxyuridine (BrdUrd) when given in an alternating weekly fashion with radiation therapy for patients with pancreatic cancer. Methods and Materials: Patients with resected or locally unresectable pancreatic cancer were eligible if distant metastases were not present. A continuous intravenous infusion of BrdUrd was given on weeks 1, 3, 5, and 7. Twice a day radiation therapy (1.5 Gy per fraction) was given on weeks 2, 4, 6, and 8 to the pancreas/pancreatic bed (total dose 60 Gy) and draining regional lymph nodes (total dose 45 Gy). The starting dose of BrdUrd was 800 mg/m2/day with a planned escalation to 1000 mg/m2/day if at least six out of eight patients were without Grade ≥ 3 toxicity. Patients were assessed weekly for toxicity, and were followed every 3 months after treatment for complications and survival. Results: Fifteen patients with resected (six) or unresectable (nine) pancreatic cancer were enrolled. One patient failed to complete therapy due to tumor progression. One of 11 patients treated with 800 mg/m2/day had a Grade 3 toxicity, while Grade 3 or 4 toxicity was found in all 3 patients receiving 1000 mg/m2/day. The dose-limiting toxicities were hematologic. The acute gastrointestinal toxicity was minimal. Two patients, including one with unresectable disease, were without evidence of disease during exploration for complications (ulcer, small bowel obstruction). Conclusions: The recommended dose of BrdUrd for Phase II study is 800 mg/m2/day. The gastrointestinal mucosa did not appear to be sensitized by this method of BrdUrd administration. The presence of a pathologic complete response is encouraging. Further improvements in radiosensitization are possible and may lead to improved local control

  20. Radiation therapy and bromodeoxyuridine chemotherapy followed by procarbazine, lomustine, and vincristine for the treatment of anaplastic gliomas

    International Nuclear Information System (INIS)

    Purpose: To conduct a Phase II study to evaluate the long-term efficacy and safety of radiotherapy combined with intravenous bromodeoxyuridine for patients with anaplastic glioma tumors. Methods and Materials: Between 1983 and 1987, study patients received 1.7-1.8 Gy radiation once a day, Monday through Friday, to a total dose of 60 Gy. On the Thursday prior to beginning radiotherapy and for the next 5 weeks (6 weeks total), patients received a continuous 96 h intravenous infusion of bromodeoxyuridine at 0.8 g/m2/24 h; following radiotherapy, patients received procarbazine, lomustine (CCNU), and vincristine (PCV) for 1 year or until tumor progressed. Results: One-hundred thirty eight patients (median age, 43 years) were evaluable for analysis. Estimated 4-year survival for the anaplastic astrocytoma (AA) stratum (n 116) is 46%. For the astrocytoma (ASTRO) stratum (n = 22), the 6-year survival is estimated at 79%. Estimated 4-year progression-free survival for AAs is 42%, and for ASTROs, 68%. Whole brain irradiation was used in 23% and limited-field irradiation in 77%; patients receiving limited-field irradiation had a better survival rate (p = 0.07). Total tumor resection was performed in 15%, partial resection in 53%, and biopsy only in 32%. For the 81 patients with tumor recurrence, 34 (42%) are known to have received additional treatment(s). For AA, fits of the Cox proportional hazards regression model showed that covariates individually predictive of survival were younger age (p < 0.001), Karnofsky performance score (p = 0.04), and extent of surgery (p = 0.04); limited-field irradiation was not significant (p = 0.10). Major toxicities were rash during Weeks 1 through 6 requiring dose modification in 14%, Grade ≥III leukopenia in 18%, and Grade ≥III thrombocytopeni in 9%. Conclusion: The study suggests that the bromodeoxyuridine-radiotherapy-PCV, compared with other published therapies, can improve progression-free survival, and aggressive treatment of ASTRO

  1. Detection of viral genomes in the liver by in situ hybridization using 35S-, bromodeoxyuridine-, and biotin-labeled probes

    International Nuclear Information System (INIS)

    Methods employing 35S-, biotin-, and bromodeoxyuridine (BrdUrd)-labeled DNA probes were compared for the detection of hepatitis B virus (HBV) and cytomegalovirus (CMV) in the liver. The results demonstrate that: 1) HBV can be detected reliably only by the use of radiolabeled probes, whereas methods employing nonradioactive probes obviously are not sensitive enough for this virus. The use of 35S-labeled probes shortens the exposure times considerably in comparison to tritiated probes. 2) Biotin-labeled probes are of limited value for in situ hybridization on liver tissues because the presence of endogenous avidin-binding activity often leads to false positive results. 3) Brd-Urd-labeled probes are a useful alternative to biotinylated probes for the detection of CMV. In comparison with biotinylated probes, BrdUrd-labeled probes produce a specific signal of similar staining intensity in the absence of background staining in the liver

  2. Induction (or stimulatin) of prolactin and growth hormone production in a rat pituitary tumor cell line by bromodeoxyuridine

    International Nuclear Information System (INIS)

    Under basal conditions, a rat pituitary tumor cell line (C3 11RAP) does not secrete any detectable PRL, FSH, and LH, and secretes only minute amounts of GH (27.1 +/- 0.5 ng/106 cells . 24 h), as evaluated by RIA. Bromodeoxyuridine (BrdUrd) added to the culture medium induced the accumulation of PRL into cells and medium, increased that of GH, but did not induce that of LH or FSH. The amount of radioimmunoassayable PRL and GH accumulated in the medium, increased after a lag period of 15 days and was drug concentration dependent. Maximal accumulation was 232.9 +/- 36.8 and 493.6 +/- 41.5 ng/106 cells . 24 h for PRL and GH, respectively, at 50 μg/ml BrdUrd. In the presence of BrdUrd (greater than or equal to20 μg/ml), the cells grew more slowly and were more strongly attached to the flasks. All of the effects induced by BrdUrd were reversible. PRL and GH were characterized by three methods: 1) radiocompetition with increasing dilution of samples; 2) Sephadex chromatography, followed by RIAs; and 3) sodium dodecyl sulfate-polyacrylamide gel electrophoresis done on the immunoprecipitate of the proteins secreted by cells incubated with [3H]leucine. Chronic treatment with TRH (3 x 10-6 M) of cells grown without BrdUrd was unable to increase the production of GH or to induce that of PRL. On the other hand, after the same treatment of cells cultured in the presence of BrdUrd, the amounts of PRL accumulated in the culture medium or cells were increased 2- to 7-fold over unstimulated levels; under the same conditions, GH accumulation in the medium was also increased, but this augmentation was less than that of PRL

  3. A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

    Directory of Open Access Journals (Sweden)

    Mauricas Mykolas

    2005-09-01

    Full Text Available Abstract Background Presently available flow cytometric methods of bromodeoxyuridine (BrdUrd labelling do not provide information on the cell cycle time (TC and the growth fraction (GF. In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling. Methods The proposed method is based on two assumptions: (1 the number of labelled cells traversing the cell cycle per unit time is constant and (2 the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture. Results The suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 ± 1.1 h and 21.6 ± 0.9 h, respectively. GF in tumours at day 10 was lower than GF at day 4 (54.2 ± 7.7% vs. 79.2 ± 5.9%, p = 0.0003. Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively. Conclusion The proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed

  4. The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions

    Science.gov (United States)

    Ligasová, Anna; Liboska, Radek; Rosenberg, Ivan; Koberna, Karel

    2015-01-01

    We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority. PMID:26161977

  5. Estimation of S phase duration in goat epidermis by an in vivo intradermal double labelling technique using bromodeoxyuridine and tritiated thymidine

    International Nuclear Information System (INIS)

    When studying skin diseases resulting from alterations in the rate of epidermal cell turnover it is useful to be able to quantify parts of the epidermal cell cycle. An in vivo intradermal technique is described which uses tritiated thymidine followed by bromodeoxyuridine to label cells in the S phase of the cell cycle. Combined autoradiographic and immunocytochemical techniques were used to quantify the flux of cells into and out of S phase. These results were then used to estimate the length of S phase. The technique was found to provide clear distinctive labelling of S phase nuclei with both reagents. This avoided many of the problems encountered with double labelling techniques using two radioactive labels. S phase was calculated to be 7.7 hours for goat skin

  6. Effect of 3-aminobenzamide on the cell cycle in x-irradiated Chinese hamster cells replicating in medium containing 5-bromodeoxyuridine

    International Nuclear Information System (INIS)

    A specific inhibitor of poly(ADP-ribose)polymerase- 3 -aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-Kl cells, cultivated in medium containing 5-bromodeoxyuridine (10 mkM, BDU-cells), and increase their radiosensitivity; 2) induce G2-delay in BDU-cells while progressing through the cell cycle as analysed by the DNA flow cytometry; 3) increase to a great degree G2-delay in X-irradiated BDU-cells. 3-Aminobenzamide is primarily effective when it is present during the first or two first cell cycles after the initial addition of BDU. The above data confirm the involvement, presumably an indirect one, of ADP-ribosylation in the DNA repair through affecting the chromatin structure

  7. Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation

    International Nuclear Information System (INIS)

    Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs. The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts

  8. Effect of bromodeoxyuridine on radiation-induced DNA damage and repair based on DNA fragment size using pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    We have used biphasic linear ramping pulsed-field gel electrophoresis (PFGE) to understand the effect of incorporation of bromodeoxyuridine (BrdUrd) on radiation-induced DNA damage and repair. This technique permits a determination of the fragment size distribution produced immediately after irradiation as well as during the repair period. We found that incorporation of BrdUrd increased the induction and decreased the repair of radiation damage. The fragment size distribution was consistent with a random breakage model. When we found that significantly more damage was detected after irradiation of deproteinized DNA compared to intact cells, we studied the effects of BrdUrd incorporation on the radiation response of cells or DNA at various phases of preparation for electrophoresis: cells adherent to the culture dish (A), trypsinized cells (B), agarose-embedded cells (C) and deproteinized DNA (D). Although there was a general tendency to detect more damage when irradiation was performed later in the preparation process, steps B and C were the only successive steps which were significantly different. These findings demonstrate that incorporation of BrdUrd randomly increases the induction of radiation damage and decreases its repair at the level of 200 kbp to 5 Mbp fragments. Furthermore, they confirm that the amount of damage detected depends upon the conditions of the cells or DNA at the time of irradiation. 34 refs., 5 figs., 2 tabs

  9. Radiosensitization of hematopoietic precursor cells (CFU/sub c/) in glioblastoma patients receiving intermittent intravenous infusions of bromodeoxyuridine (BUdR)

    International Nuclear Information System (INIS)

    The potential use of bromodeoxyuridine (BUdR) as a radiosensitizer given by an intermittent intravenous route is being studied in a Phase I/II trial at the Naitonal Cancer Institute. In order to assess the extent of radiosensitization, we have studied the radiation response of human bone marrow cells CFUc taken form 6 patients prior to and after a 14-day infusion of BUdR. Varying concentrations (1000-1500 mg) of BUdR were infused for 12 hours 12 hours every 24 hours for up to 14 consecutive days. Cells survival was determined by colony formation of CFUc in soft agar suspensions. X ray survival curves were generated over a dose range of 0-300 rad and the slopes of the survival curves (D0) before and after BUdR infusion were compared. Radiation enhancement ratios (ER)(D0 per-BUdR/D0 post-BUdR) ranged form 1.0-2.2 and appeared to be BUdR dose dependent. Above 650 mg/m2/12 hours is well tolerated and may result in radiosensitizaiton of CFUc in man

  10. Mineral Type and Solution Chemistry Affect the Structure and Composition of Actively Growing Bacterial Communities as Revealed by Bromodeoxyuridine Immunocapture and 16S rRNA Pyrosequencing.

    Science.gov (United States)

    Kelly, L C; Colin, Y; Turpault, M-P; Uroz, S

    2016-08-01

    Understanding how minerals affect bacterial communities and their in situ activities in relation to environmental conditions are central issues in soil microbial ecology, as minerals represent essential reservoirs of inorganic nutrients for the biosphere. To determine the impact of mineral type and solution chemistry on soil bacterial communities, we compared the diversity, composition, and functional abilities of a soil bacterial community incubated in presence/absence of different mineral types (apatite, biotite, obsidian). Microcosms were prepared containing different liquid culture media devoid of particular essential nutrients, the nutrients provided only in the introduced minerals and therefore only available to the microbial community through mineral dissolution by biotic and/or abiotic processes. By combining functional screening of bacterial isolates and community analysis by bromodeoxyuridine DNA immunocapture and 16S rRNA gene pyrosequencing, we demonstrated that bacterial communities were mainly impacted by the solution chemistry at the taxonomic level and by the mineral type at the functional level. Metabolically active bacterial communities varied with solution chemistry and mineral type. Burkholderia were significantly enriched in the obsidian treatment compared to the biotite treatment and were the most effective isolates at solubilizing phosphorous or mobilizing iron, in all the treatments. A detailed analysis revealed that the 16S rRNA gene sequences of the OTUs or isolated strains assigned as Burkholderia in our study showed high homology with effective mineral-weathering bacteria previously recovered from the same experimental site. PMID:27138048

  11. Outcome and human epidermal growth factor receptor (HER) 1–4 status in invasive breast carcinomas with proliferation indices evaluated by bromodeoxyuridine labelling

    International Nuclear Information System (INIS)

    We have shown previously that whereas overexpression of human epidermal growth factor receptor (HER)1, HER2 and HER3 is associated with poor prognosis in breast cancer, HER4 is associated with a good prognosis. Cell proliferation is a key component of aggressive cancers and is driven by growth factors. In this study, bromodeoxyuridine (BrdU)-derived proliferation indices are correlated with clinical outcome and HER1–4 status for further clarification of the differing roles for the HER family at a biological level. Seventy-eight invasive breast cancers had BrdU labelling in vivo to determine the BrdU labelling index (BLI) and the potential tumour doubling time (Tpot). Long-term clinical follow-up was available for these patients. We used immunohistochemistry to establish the HER1–4 status in 55 patients from the BrdU cohort. We demonstrate a significant correlation between high BLI values and breast cancer-specific death (P = 0.0174). Low Tpot times were also significantly correlated with breast cancer-specific death (P = 0.0258). However, BLI did not independently predict survival in Cox's multiple regression analysis when combined with other prognostic factors such as size, grade and nodal status. Tumours found to be positive for HER1, HER2 or HER3 had significantly (P = 0.041) higher labelling indices, with HER1 also showing significantly higher indices when considered independently (P = 0.024). Conversely, HER4 positivity was significantly correlated (P = 0.013) with low BLI values, in line with previous data associating this receptor with good prognosis tumours. These results support the hypothesis that HER1–3 are associated with driving tumour proliferation, whereas HER4 is involved in a non-proliferative or even protective role

  12. Bromodeoxyuridine (BrdU) treatment to measure hepatocellular proliferation does not mask furan-induced gene expression changes in mouse liver

    International Nuclear Information System (INIS)

    Bromodeoxyuridine (BrdU) is a synthetic nucleoside used to detect cellular proliferation. BrdU incorporates in the place of thymine but pairs with guanine, thereby increasing the risk of transition mutations in dividing cells. Given its mutagenicity, standard practice is to use a second cohort of animals for parallel toxicogenomics studies; however, the impact of BrdU on global gene expression is unknown. To test this, we performed a case study to determine whether the molecular mode of action of furan, a liver carcinogen, could be detected in BrdU-treated samples. We measure global hepatic gene expression using Agilent DNA microarrays in female B6C3F1 mice that were sub-chronically exposed to 0, 1, 4, or 8 mg/kg bodyweight (bw) per day furan either in the presence (+BrdU) or absence (−BrdU) of BrdU. Exposure to 0.02% BrdU in drinking water for five days resulted in minimal gene expression changes. A comparison of +BrdU versus −BrdU control mice revealed only 11 probes with fold change ≥ 1.5 and false discovery rate (FDR) corrected p ≤ 0.05. The same comparison in the high dose group yielded only 3 differentially expressed probes. Differentially expressed gene lists generated for furan-treated versus control mice and were compared for the −BrdU and +BrdU groups. The high dose of furan had 452 shared probes and 27 and 90 unique probes for −BrdU and +BrdU groups, respectively. These differences did not impact hierarchical clustering. Further, they did not impair detection of the previously reported furan mode of action, which was well represented in the BrdU-treated samples. Taken together, we demonstrate that BrdU treatment does not mask important furan-induced transcriptional changes. We suggest that BrdU-treated mice could be used for toxicogenomic analysis, which would generally halve the number of rodents required for toxicogenomics studies. However, we also recommend that this type of case study be repeated for other chemicals before the use of Brd

  13. Results of a randomized comparison of radiotherapy and bromodeoxyuridine with radiotherapy alone for brain metastases: report of RTOG trial 89-05

    International Nuclear Information System (INIS)

    Purpose: To determine if the addition of bromodeoxyuridine (BrdUrd) to radiotherapy prolongs survival when compared to radiotherapy alone in patients with brain metastases. Methods and Materials: Seventy-two patients with brain metastases were randomized to 37.5 Gy in 15 fractions of 2.5 Gy or to the same dose with BrdUrd 0.8 g/m2 per day for 4 days of each of 3 weeks. Drug treatment was begun on Thursday or Friday before the first week of radiotherapy. Patients had a Karnofsky performance score of at least 70, a neurological function classification of 1 or 2, and any primary tumor except central nervous system (CNS), leukemia, or lymphoma. The primary was absent, controlled, or under active radiotherapy. Patients were free of other metastases. They were stratified by primary site (breast, lung or other), number of metastases (single or multiple) and age ( 60). Results: There was no significant difference between the two treatment arms (p 0.904). The study was open from October 1989 to March 1993 and accrued 72 patients. Only one patient in the RT only arm remains alive. The two treatment arms were balanced with respect to all stratification variables. Toxicity due to radiotherapy was similar in both arms. BrdUrd caused significant Grade 4 and 5 hematologic and skin toxicity in five patients. Two patients died due to hematologic toxicity and one from a Stevens-Johnson type skin reaction. Phenytoin played a role in the skin reactions and ranitidine was associated with the hematologic deaths. Ranitidine was eliminated, BrdUrd was discontinued after any hematologic toxicity, and no further Grade 4 or 5 toxicities were seen. The median survival was 6.12 months in the radiotherapy group and 4.3 in the BrdUrd group (p = 0.904). Patients with solitary brain metastases had significantly better survival (p = 0.031). Conclusions: BrdUrd did not enhance the efficacy of the radiotherapy regimen tested, in spite of the fact that brain metastases have shown high labeling indices

  14. Utilisation de l'essai comete et du biomarqueur gamma-H2AX pour detecter les dommages induits a l'ADN cellulaire par le 5-bromodeoxyuridine post-irradiation

    Science.gov (United States)

    La Madeleine, Carole

    Ce memoire est presente a la Faculte de medecine et des sciences de la sante de l'Universite de Sherbrooke en vue de l'obtention du grade de maitre es sciences (M.Sc.) en radiobiologie (2009). Un jury a revise les informations contenues dans ce memoire. Il etait compose de professeurs de la Faculte de medecine et des sciences de la sante soit : Darel Hunting PhD, directeur de recherche (departement de medecine nucleaire et radiobiologie), Leon Sanche PhD, directeur de recherche (departement de medecine nucleaire et radiobiologie), Richard Wagner PhD, membre du programme (departement de medecine nucleaire et radiobiologie) et Guylain Boissonneault PhD, membre exterieur au programme (departement de biochimie). Le 5-bromodeoxyuridine (BrdU), un analogue halogene de la thymidine reconnu depuis les annees 60 comme etant un excellent radiosensibilisateur. L'hypothese la plus repandue au sujet de l'effet radio sensibilisant du BrdU est qu'il augmente le nombre de cassures simple et double brin lorsqu'il est incorpore dans l'ADN de la cellule et expose aux radiations ionisantes. Toutefois, de nouvelles recherches semblent remettre en question les observations precedentes. Ces dernieres etudes ont confirme que le BrdU est un bon radiosensibilisateur, car il augmente les dommages radio-induits dans l'ADN. Mais, c'est en etant incorpore dans une region simple brin que le BrdU radiosensibilise l'ADN. Ces recherches ont egalement revele pour la premiere fois un nouveau type de dommages produits lors de l'irradiation de l'ADN contenant du BrdU : les dimeres interbrins. Le but de ces travaux de recherche est de determiner si la presence de bromodeoxyuridine dans l'ADN augmente l'induction de bris simple et / ou double brin chez les cellules irradiees en utilisant de nouvelles techniques plus sensibles et specifiques que celles utilisees auparavant. Pour ce faire, les essais cometes et la detection des foci H2AX phosphorylee pourraient permettre d'etablir les effets engendres par

  15. Evaluation of the radiosensitizing to treatment with 153Sm-EDTMP, of haematopoietic cells of the bone marrow by means of bromodeoxyuridine incorporation into DNA, in a murine model

    International Nuclear Information System (INIS)

    Bromodeoxyuridine (BrdU) has been shown to have a radiosensitizing effect, and its incorporation into DNA prior to administration of a bone-seeking radiopharmaceutical could increase the efficiency of bone marrow ablation, and even increase the specificity of radiation exposure for therapeutic purposes. The aim of the present study was to determine the effect of BrdU incorporation into DNA on the genotoxic and cytotoxic effects of samarium-153 ethylenediaminetetra-methylene phosphonate (153Sm-EDTMP) in murine bone marrow cells. BALB/c male mice (N = 5 in each experiment) were treated with one of the following substances: a) BrdU (0.25 mg/g) b) 153-EDTMP (11.5 ± 3 MBq) c) BrdU (0.25 mg/g) plus 153Sm-EDTMP (11.5 ± MBq), there was also an untreated control. Cytotoxicity and genotoxicity were established by time-response and absorbed dose-response curves of polychromatic erythrocyte (PCE) and micro nucleated polychromatic erythrocyte (MN-PCE) frequencies, respectively, in murine peripheral blood samples in vivo. The significance of the differences between groups was determined by a variation of Dunett test for multiple groups and different-sized groups of a student test. Beta-absorbed dose fractions obtained from MNCP4B Monte Carlo computer code were used for mice bone marrow dosimetry calculations. At an average radiation absorbed dose of 0.38 Gy, 0.56 Gy and 0.82 Gy at 24, 40 and 72 h respectively, cells from animals treated with 153Sm-EDTMP showed a clear and significant induction of MN-PCE after 24 h, with the maximum response at 40 h, however, cells from group treated with BrdU plus 153Sm-EDTMP paradoxically showed MN-PCE frequencies only slightly higher than the control at the same absorbed dose. Treatment with 153Sm-EDTMP caused a slight reduction in PCE frequency, but exposure to BrdU or BrdU plus 153Sm-EDTMP induced a substantial and significant reduction in PCE frequency from 32 h to the end of the experiment (72 h). The PCE frequencies in the Brd

  16. Influence of bromodeoxyuridine radiosensitization on malignant glioma patient survival: a retrospective comparison of survival data from the Northern California oncology group (NCOG) and radiation therapy oncology group trials (RTOG) for glioblastoma multiforme and anaplastic astrocytoma

    International Nuclear Information System (INIS)

    Purpose: To examine the effect of treatment using Bromodeoxyuridine (BrdU) during radiation therapy on malignant glioma patient survival by comparing historical survival data from several large clinical trials. Methods: A retrospective analysis of patient data from Radiation Therapy Oncology Group (RTOG) trials 74-01, 79-18, and 83-02 and the Northern California Oncology Group (NCOG) study 6G-82-1 was conducted. Patient data was supplied by both groups, and analyzed by the RTOG. Pretreatment characteristics including age, extent of surgery, Karnofsky Performance Status (KPS), and histopathology were collected; the only treatment variable evaluated was the use of BrdU during radiation therapy. Radiation dose, dose-fractionation schedule, use of chemotherapy, and/or type of chemotherapy was not controlled for in the analyses. Univariate and multivariate analyses were conducted to examine the potential treatment effect of BrdU on patient survival. Results: Data from 334 patients treated with BrdU on NCOG 6G-82-1 and 1743 patients treated without BrdU on 3 RTOG studies was received. Patients were excluded from the review if confirmation of eligibility could not be obtained, if the patient was ineligible for the study they entered, if central pathology review was not done, or if radiotherapy data was not available. Patients treated according to the RTOG studies had to start radiotherapy within 4 weeks of surgery; no such restriction existed for the NCOG studies. To ensure comparability between the studies, patients from the NCOG studies who began treatment longer than 40 days from surgery were also excluded. The final data set included 296 cases from the NCOG studies (89%) and 1478 cases from the RTOG studies (85%). For patients with glioblastoma multiforme (GBM) the median survival was 9.8 months in the RTOG studies and 13.0 months in the NCOG trial (p < 0.0001). For patients with AA the median survival was 35.1 months for the RTOG studies and 42.8 months in the NCOG

  17. Biochemical properties of the bromodeoxyuridine-induced guinea pig virus.

    Science.gov (United States)

    Michalides, R; Schlom, J; Dahlberg, J; Perk, K

    1975-10-01

    The biophysical and biochemical properties of the virus particles released by guinea pig embryo cells treated with 5-bromo-2'-deoxyuridine (BUdR) have been compared to those of the B-type mouse mammary tumor virus (MMTV) and the C-type Rauscher murine leukemia virus. The high-molecular-weight (60 to 70S) RNA of the BUdR-induced guinea pig virus (GPV) has a molecular weight of 8 X 106 when measred by mixed agarose polyacylamide gel electrophoresis. The virus particles isolated from the tissue culture medium of BUdR-induced guniea pig cells have the following properties in common with MMTV: (i) a buoyant density of 1.18 g/ml in sucrose and 1.21 g/ml in CsCl, and (ii) a DNA polymerase that prefers Mg2+ over Mn2+ in an assay using the synthetic template poly(rC):oligo(dG). No nucleic acid sequence homology between GPV RNA and the viral RNAs of the MMTV, murine leukemia virus, hamster sarcoma virus, or Mason-Pfizer monkey virus could be observed in a competition hybridization assay using the radioactive-labeled GPV 60 to 70S RNA. By this same competition by hybridization assay the frequency of GPV proviral sequences was estimated to be at least 83 per haploid cellular genome of guniea pig cells. No nucleic acid sequences related to be GPV RNA were detected in the DNA of normal tissues of mice, rats, cats, dogs, baboons, or humans by direct RNA-DNA hybridization using radioactive GPV60 to 70S RNA. PMID:51933

  18. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine.

    OpenAIRE

    Dolbeare, F; Gratzner, H; Pallavicini, M. G.; Gray, J.W. (Joe W.)

    1983-01-01

    We have developed a procedure for simultaneous flow cytometric measurement of cellular DNA content and amount of BrdUrd incorporated into cellular DNA. Propidium iodide was used as a fluorescent probe for total cellular DNA and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescein-labeled goat anti-mouse antibody was used to fluorescently label the bound anti-BrdUrd probe. Bivariate DNA/BrdUrd distributions measured for Chinese hamster ovary ce...

  19. Evaluation of the radiosensitizing to treatment with {sup 153}Sm-EDTMP, of haematopoietic cells of the bone marrow by means of bromodeoxyuridine incorporation into DNA, in a murine model; Evaluacion de la radiosensibilizacion al tratamiento con {sup 153}Sm-EDTMP, de las celulas hemotopoyeticas de la medula osea mediante la incorporacion de bromodesoxiuridina (BrdU) en el ADN, en un modelo murino

    Energy Technology Data Exchange (ETDEWEB)

    Morales A, E.

    2008-07-01

    Bromodeoxyuridine (BrdU) has been shown to have a radiosensitizing effect, and its incorporation into DNA prior to administration of a bone-seeking radiopharmaceutical could increase the efficiency of bone marrow ablation, and even increase the specificity of radiation exposure for therapeutic purposes. The aim of the present study was to determine the effect of BrdU incorporation into DNA on the genotoxic and cytotoxic effects of samarium-153 ethylenediaminetetra-methylene phosphonate ({sup 153}Sm-EDTMP) in murine bone marrow cells. BALB/c male mice (N = 5 in each experiment) were treated with one of the following substances: a) BrdU (0.25 mg/g) b) {sup 153}-EDTMP (11.5 +- 3 MBq) c) BrdU (0.25 mg/g) plus {sup 153}Sm-EDTMP (11.5 +- MBq), there was also an untreated control. Cytotoxicity and genotoxicity were established by time-response and absorbed dose-response curves of polychromatic erythrocyte (PCE) and micro nucleated polychromatic erythrocyte (MN-PCE) frequencies, respectively, in murine peripheral blood samples in vivo. The significance of the differences between groups was determined by a variation of Dunett test for multiple groups and different-sized groups of a student test. Beta-absorbed dose fractions obtained from MNCP4B Monte Carlo computer code were used for mice bone marrow dosimetry calculations. At an average radiation absorbed dose of 0.38 Gy, 0.56 Gy and 0.82 Gy at 24, 40 and 72 h respectively, cells from animals treated with {sup 153}Sm-EDTMP showed a clear and significant induction of MN-PCE after 24 h, with the maximum response at 40 h, however, cells from group treated with BrdU plus {sup 153}Sm-EDTMP paradoxically showed MN-PCE frequencies only slightly higher than the control at the same absorbed dose. Treatment with {sup 153}Sm-EDTMP caused a slight reduction in PCE frequency, but exposure to BrdU or BrdU plus {sup 153}Sm-EDTMP induced a substantial and significant reduction in PCE frequency from 32 h to the end of the experiment (72 h

  20. Cell proliferation in the Drosophila adult brain revealed by clonal analysis and bromodeoxyuridine labelling

    OpenAIRE

    Brand Andrea H; Egger Boris; von Trotha Jakob W

    2009-01-01

    Abstract Background The production of new neurons during adulthood and their subsequent integration into a mature central nervous system have been shown to occur in all vertebrate species examined to date. However, the situation in insects is less clear and, in particular, it has been reported that there is no proliferation in the Drosophila adult brain. Results We report here, using clonal analysis and 5'-bromo-2'-deoxyuridine (BrdU) labelling, that cell proliferation does occur in the Droso...

  1. Phenobarbital-induced hepatocellular proliferation: anti-bromodeoxyuridine and anti-proliferating cell nuclear antigen immunocytochemistry.

    Science.gov (United States)

    Jones, H B; Clarke, N A; Barrass, N C

    1993-01-01

    We report modifications to immunocytochemical detection procedures for proliferating cell nuclear antigen (PCNA) which permit its identification in liver samples previously fixed for BrdU immunocytochemistry. Both methods have been used for the assessment of phenobarbital-induced cell proliferation in rat liver. The difficulties associated with the hitherto unsuccessful application of PCNA immunocytochemical methods to tissues fixed in formalin for BrdU visualization were overcome by epitope unmasking with acid hydrolysis, extension of primary antiserum (PC10) incubation, and employment of streptavidin-ABC-HRP. BrdU delivery via osmotic minipumps for 48 hr before euthanasia, followed by fixation in cold formalin for 14 days, yielded reliable and reproducible hepatocellular labeling and a peak of cell proliferation in all lobes on Day 3 (i.e., labeling during Days 1-3) of dosing with 80 mg/kg/day phenobarbital. Labeling indices (LI) of both control and phenobarbital-treated liver were lower in the left and right median lobes as compared with the lateral lobes. In sections of the left lateral lobe from the same liver, PCNA immunocytochemistry revealed a peak of proliferative activity (about one third of the maximum LI generated by BrdU incorporation) on Day 1. These findings, together with the advantages and disadvantages of both techniques, are discussed in the context of their applications to different investigative requirements. PMID:8093255

  2. Use of the 5-bromodeoxyuridine-labelling technique for exploring mechanisms involved in the formation of chromosomal aberrations. Pt. 2

    International Nuclear Information System (INIS)

    Synchronized G1 CHO cells with chromosomes of TB or TT constitution were irradiated with X-rays, short-wave UV and long-wave UV. The types and frequencies of chromosomal aberrations observed in the ensuing mitosis were studied. X-Rays induced predominantly chromosome types of aberration in chromosomes of TT constitution, whereas both chromosome- and chromatid-types of aberration were induced in cells with chromosomes of TB constitution. Short-wave UV induced only chromatid types of aberration in cells containing chromosomes of TT constitution, but both chromosome and chromatid types of aberration in cells with chromosomes of TB constitution. Long-wave UV induced chromosome and chromatid types of aberration in cells with chromosomes of TB constitution and no aberrations in cells containing chromosomes of TT constitution. Long-wave UV-irradiation of cells containing chromosomes of TB constitution increases the frequencies of SCEs. The relationship between chromosome constitution (TT or TB), the type of lesions induced by the 3 different agents employed, and the types chromosomal aberration induced are discussed. (orig.)

  3. Enhancement of chromosome aberrations induced in 5'-bromodeoxyuridine-labelled Chinese hamster ovary cells by monochromatic synchrotron radiations

    International Nuclear Information System (INIS)

    The chromosome study for the radiation-induced enhancement in producing various types of aberrations was conducted in a cultured cell line of CHO (Chinese hamster ovary) cells that were pre-labeled with BUdR (5'-deoxybromouridine) and irradiated by monochromatic X-rays with a slightly shorter wavelength (0.9 A) than the K adsorption edge of bromine (0.92 A). The dose-response changes in terms of frequencies of single-arm breaks and isochromatid breaks have shown that a maximum effect was produced by the combination of 0.9-A wavelength of X-irradiation and Br-incorporation and was above the magnitude of the so-called brome-induced sensitization obtained with 1.0-A wavelength X-rays. Such additional effect in the bromine-sensitized cells may be interpreted as the biological effect due to Auger electrons produced by photoelectric stimulation with 0.9-A monochromatic X-rays

  4. The influence of bromodeoxyuridine on the induction and repair of DNA double-strand breaks in glioblastoma cells

    International Nuclear Information System (INIS)

    Aims: To examine the dose response of DNA damage and its modification by the radiosensitizer, 5-bromo-2'-deoxyuridine (BrdU). The sensitizing mechanism is analyzed with regard to its influence on the induction and repair of DNA double-strand breaks (DSBs). Material and Methods: Cells from three different human glioblastoma lines, A7, LH and U87MG, were X-irradiated with and without exposure to BrdU. DNA fragments were separated by field-inversion gel electrophoresis (FIGE) and quantified by fluorometry immediately and 24 h after irradiation. Results: In all cell lines, the dose response followed a linear-quadratic rather than a purely linear function. BrdU-treated cells exhibited a significantly higher amount of mobile DNA. In repair experiments with and without BrdU, the amount of mobile DNA fell close to control values within 24 h. Conclusions: The linear-quadratic model appropriately describes the X-ray induced fragmentation of DNA. BrdU sensitizing acts predominantly by increasing DNA fragility, and not by impairing damage repair. The amount of DSBs persistent after 24 h of repair is minimal, even after highly cytotoxic doses. However, it appears to depend on the extent of initial damage, causing sensitized cells to retain more DSBs than unsensitized cells. (orig.)

  5. The tumour distribution of bromodeoxyuridine labelled S-phase cells is found to be strongly dose dependant

    International Nuclear Information System (INIS)

    Bromodeoxyurdine (BrdU) is used extensively to measure the fraction of S-phase cells in tumours. Unlike endogenous markers of proliferation, such as PCNA and Ki-67, BrdU is exogenously administered and reaches the tumour via vasculature where it must then distribute throughout the tissue in order to label S-phase cells. Interest in using BrdU labelling of histological sections to evaluate the distribution of the effect of different treatment modalities on tumours led us to study the ability of BrdU to distribute within tissue. The study used SiHa (human cervix squamous cell carcinoma) xenografts, a tumour that exhibits cords of cells extending up to 150 μm away from blood vessels. A quantitative microscopy-based technique was employed to determine the distribution of S-phase labelled cells relative to the vasculature over a dose range of 25-2000 mg/kg BrdU. Detection of BrdU incorporation in DNA was carried out immunohistochemically and vasculature was identified using perfusion of carbocyanine, a fluorescent perivascular stain. Analysis of BrdU labelling distribution in the tissue found that a dose of 1000 mg/kg was required to label cells furthest from vasculature. Dosing at lower levels resulted in only the cells close to blood vessels being labelled. This result is surprising since 100 mg/kg BrdU is commonly used in flow cytometry studies. Results were compared with penetration seen in vitro using multilayered cell culture, a three-dimensional tissue culture model of solid tumours. Using multilayered cell culture, an exposure of 100 μM BrdU for 1 hour was required for labelling of S-phase cells 150 μm into the tissue, while cells adjacent to the edge of the tissue could be adequately labelled with just 5 μM BrdU for 1 hour. The AUC for a 100 mg/kg BrdU dose in mice was found to be ∼30 μM x h

  6. E-cadherin expression and bromodeoxyuridine incorporation during development of ovarian inclusion cysts in age-matched breeder and incessantly ovulated CD-1 mice

    Directory of Open Access Journals (Sweden)

    Beaugié Clare R

    2007-04-01

    Full Text Available Abstract Background Female CD-1/Swiss Webster mice subjected to incessant ovulation for 8 months and 12-month breeder mice both developed ovarian inclusion cysts similar to serous cystadenomas. The majority of cysts appeared to be dilated rete ovarii tubules, but high ovulation number resulted in more cortical inclusion cysts. We hypothesized that comparison of inclusion cyst pathology in animals of the same age, but with differences in total lifetime ovulation number, might allow us to determine distinguishing characteristics of the two types of cyst. Methods Ovaries from breeder mice (BR or females subjected to incessant ovulation (IO were compared at 6-, 9- and 12-months of age. Ovaries were serially sectioned and cysts characterized with regard to location and histology, E-cadherin immunoreactivity and rates of BrdU incorporation. Results Inclusion cysts developed with age in BR and IO ovaries. The majority of cysts were connected to the ovarian hilus. Two cortical inclusion cysts were observed in ten IO ovaries and one in ten BR ovaries. Low or no E-cadherin immuno-staining was seen in the OSE of all mice studied. Conversely, strong membrane immuno-staining was observed in rete ovarii epithelial cells. Variable E-cadherin immunoreactivity was seen in cells of hilar inclusion cysts, with strong staining observed in cuboidal ciliated cells and little or no staining in flat epithelial cells. Two of the three cortical cysts contained papillae, which showed E-cadherin immuno-staining at the edge of cells. However hilar and cortical cysts were not distinguishable by morphology, cell type or E-cadherin immunoreactivity. BrdU incorporation in cyst cells (1.4% [95% CI: 1.0 to 2.1] was greater than in OSE (0.7% [95% CI: 0.4 to 1.2] and very few BrdU-labeled cells were observed in rete ovarii at any age. Incessant ovulation significantly increased BrdU incorporation in OSE of older animals. Conclusion These experiments confirm ovarian inclusion cysts develop with age in the CD-1 mouse strain, irrespective of total ovulation burden. We conclude longer periods of incessant ovulation do not lead to significant changes in inclusion cyst formation or steroidogenesis in CD-1 mice and inclusion cyst type can not be distinguished by morphology, cell proliferation rate or E-cadherin immunoreactivity.

  7. The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2 '-Deoxyuridine in Cellular DNA under Various Conditions

    Czech Academy of Sciences Publication Activity Database

    Ligasová, A.; Liboska, Radek; Rosenberg, Ivan; Koberna, K.

    2015-01-01

    Roč. 10, č. 7 (2015), e0132393/1-e0132393/16. E-ISSN 1932-6203 R&D Projects: GA TA ČR TA03010598; GA TA ČR TA03010719; GA MŠk(CZ) LO1304; GA MZd NV15-31604A Grant ostatní: GA TA ČR(CZ) TE02000058 Institutional support: RVO:61388963 Keywords : anti-halodeoxyuridine antibodies * affinity assay * DNA Subject RIV: CE - Biochemistry Impact factor: 3.234, year: 2014 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132393

  8. BrdU掺入法评估化学物皮肤致敏性的实验研究%Skin sensitization test of chemicals using bromodeoxyuridine incorporation immunoassay

    Institute of Scientific and Technical Information of China (English)

    何国群; 黄俊明; 黄琼; 郑穗生; 李庆; 谭小华; 古梅英; 谢晓平

    2007-01-01

    目的 探讨BrdU掺入法在检测和评价化学物对小鼠皮肤接触致敏性的应用.方法 选择24只雌性BALB/C小鼠随机分为6组,受试物为2,4-二硝基氯苯(DNCB)和三氯乙烯(TCE),对照组受试物为AOO溶剂.将受试物连续3d涂抹于小鼠双耳背,第4天腹腔注射BrdU标记液,第5天摘取小鼠耳部淋巴结,称重,然后制备淋巴细胞悬液,用ELISA检测技术测定BrdU掺入淋巴细胞的水平,将实验组的小鼠耳部淋巴结重量和BrdU标记指数分别与对照组进行方差分析比较.结果 溶剂对照组的小鼠淋巴结重量为(0.0060±0.0015)g,0.10、0.25、0.50、1.00%浓度的DNCB实验组的淋巴结重量为(0.0120±0.0014)、(0.0137±0.0006)、(0.0156±0.0017)、(0.0241±0.0035)g,20%TCE实验组的小鼠淋巴结重量为(0.0155±0.0017)g;溶剂对照组的BrdU标记指数为0.157±0.023;各浓度的DNCB实验组的BrdU标记指数分别为0.349±0.009、0.412±0.013、0.445±0.009、0.536±0.026;20%TCE实验组的BrdU标记指数为0.433±0.025.DNCB各浓度组、TCE组的淋巴结重量和BrdU标记指数与对照组比较差异均有统计学意义(均P<0.01).结论 BrdU掺入法作为一种改良的局部淋巴结实验方法,它具有评估化学物皮肤接触致敏性的能力.

  9. A new quantitative test method for cell proliferation based on detection of the Ki-67 protein

    NARCIS (Netherlands)

    Klein, CL; Wagner, M; Kirkpatrick, CJ; Van Kooten, TG

    1999-01-01

    A quantitative method to assess cell proliferation is one essential prerequisite for testing biomaterial cytocompatibility in vitro. Currently used methods, e.g. bromodeoxyuridine incorporation, show serious disadvantages concerning either sensitivity, specificity or handling. A new enzyme linked im

  10. Turnover of T cells in murine gammaherpesvirus 68-infected mice

    DEFF Research Database (Denmark)

    Hamilton-Easton, A M; Christensen, Jan Pravsgaard; Doherty, P C

    1999-01-01

    Respiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found to ...

  11. Tumour radiosensitization with the halogenated pyrimidines 5'-bromo-and 5'-iododeoxyuridine

    Energy Technology Data Exchange (ETDEWEB)

    Epstein, A.H.; Cook, J.A.; Goffman, T. (National Cancer Inst., Bethesda, MD (United States)); Glatstein, E. (Texas Univ., Dallas, TX (United States). Southwestern Medical Center)

    1993-02-01

    The authors review studies of the use of iododeoxyuridine (IdUrd) and bromodeoxyuridine as radiosensitizers and attempt to correlate the clinical outcome for patients treated with radiation and IdUrd with the extent of halogenated pyrimidine cellular uptake and incorporation. (U.K.).

  12. Tumour radiosensitization with the halogenated pyrimidines 5'-bromo-and 5'-iododeoxyuridine

    International Nuclear Information System (INIS)

    The authors review studies of the use of iododeoxyuridine (IdUrd) and bromodeoxyuridine as radiosensitizers and attempt to correlate the clinical outcome for patients treated with radiation and IdUrd with the extent of halogenated pyrimidine cellular uptake and incorporation. (U.K.)

  13. Lamotrigine increases the number of BrdU-labeled cellsinthe rat hippocampus

    DEFF Research Database (Denmark)

    Kondziella, Daniel; Strandberg, Joakim; Lindquist, Catarina; Asztely, Fredrik

    2010-01-01

    newborn cells inrat hippocampus. Rats (on day P21) received lamotrigine, valproate, or saline intraperitoneally once dailyfor 7 days. All animals received four intraperitoneal injections of bromodeoxyuridine (BrdU) on day P28 andwere sacrificed the next day. Quantification of BrdU-labeled cells in the...

  14. Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats

    DEFF Research Database (Denmark)

    Jensen, P O; Mortensen, B T; Hodgkiss, R J; Iversen, P O; Christensen, I J; Helledie, N; Larsen, J K

    2000-01-01

    mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the...

  15. Effects of NOS inhibitor on dentate gyrus neurogenesis after diffuse brain injury in the adult rats

    Institute of Scientific and Technical Information of China (English)

    SunLi-Sha; XuJiang-ping

    2004-01-01

    Objective To investigate the effects of selective nitric oxide synthase (NOS) inhibitors on dentate gyrus neurogenesis after diffuse brain injury (DBI) in the adult rat brain. Methods Adult male SD rats were subjected to diffuse brain injury (DBI) model. By using systemic bromodeoxyuridine (BrdU) to label dividing cells, we compared the proliferation rate of

  16. Postembryonic neurogenesis in the procerebrum of the terrestrial snail, Helix lucorum L

    Science.gov (United States)

    Zakharov, I. S.; Hayes, N. L.; Ierusalimsky, V. N.; Nowakowski, R. S.; Balaban, P. M.

    1998-01-01

    Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70-80%) of the snail procerebrum are produced during the first 1-2 months of posthatching development.

  17. Cell proliferation in Helicobacter pylori associated gastritis and the effect of eradication therapy.

    OpenAIRE

    Lynch, D A; Mapstone, N P; Clarke, A. M.; Sobala, G M; Jackson, P.; Morrison, L.; Dixon, M F; Quirke, P; Axon, A T

    1995-01-01

    Helicobacter pylori causes chronic (type B) gastritis. The 'intestinal' form of gastric cancer arises against a background of chronic gastritis, and prospective epidemiological studies have shown that H pylori is a major risk factor for this. An increase in mucosal cell proliferation increases the likelihood of a neoplastic clone of epithelial cells emerging where there is chronic epithelial cell injury associated with H pylori gastritis. In vitro bromodeoxyuridine labelling of endoscopic ant...

  18. Cerebrolysin enhances neurogenesis in the ischemic brain and improves functional outcome after stroke

    OpenAIRE

    Zhang, Chunling; Chopp, Michael; Cui, Yisheng; Wang, Lei; Zhang, Ruilan; Zhang, Li; Lu, Mei; Szalad, Alexandra; Doppler, Edith; Hitzl, Monika; Zhang, Zheng Gang

    2010-01-01

    Cerebrolysin is a peptide preparation mimicking the action of neurotrophic factors and has beneficial effects on neurodegenerative diseases and stroke. The present study investigated the effect of Cerebrolysin on neurogenesis in a rat model of embolic middle cerebral artery occlusion (MCAo). Treatment with Cerebrolysin at doses of 2.5 and 5 ml/kg significantly increased the number of bromodeoxyuridine positive (BrdU+) subventricular zone (SVZ) neural progenitor cells and doublecortin (DCX) im...

  19. Exercise Enhances Learning and Hippocampal Neurogenesis in Aged Mice

    OpenAIRE

    van Praag, Henriette; Shubert, Tiffany; Zhao, Chunmei; GAGE, FRED H.

    2005-01-01

    Aging causes changes in the hippocampus that may lead to cognitive decline in older adults. In young animals, exercise increases hippocampal neurogenesis and improves learning. We investigated whether voluntary wheel running would benefit mice that were sedentary until 19 months of age. Specifically, young and aged mice were housed with or without a running wheel and injected with bromodeoxyuridine or retrovirus to label newborn cells. After 1 month, learning was tested in the Morris water ma...

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

    OpenAIRE

    Cristina Rossi; Pier Paolo Danieli; Bruno Ronchi

    2014-01-01

    The β-hexachlorocyclohexane (β-HCH) is a very stable and accumulable isomer of Lindane, a well known organochlorine pesticide. The HCHs were banned in all developed countries but to date high concern still exists for environment, animal and human health due to contaminated sites. In this study, several in vitro tests [cell viability (XTT), trypan blue exclusion (TBE), lactate dehydrogenase release (LDH) and bromodeoxyuridine (BrdU) incorporation assays] were performed to investigate the toxic...

  1. Mass spectral characterization of a protein-nucleic acid photocrosslink.

    OpenAIRE

    Golden, M. C.; Resing, K. A.; Collins, B. D.; Willis, M. C.; Koch, T H

    1999-01-01

    A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with ...

  2. Immunomodulatory effects of Potentilla indica and Dendrophthoe pentandra on mice splenocytes and thymocytes

    OpenAIRE

    Ang, Hui Ying; SUBRAMANI, TAMILSELVAN; Yeap, Swee Keong; OMAR, ABDUL RAHMAN; HO, WAN YANG; Abdullah, Mohd Puad; Alitheen, Noorjahan Banu

    2014-01-01

    Immunomodulators are agents that are able to stimulate or inhibit the immune response. The leaf extracts from Potentilla indica and Dendrophthoe pentandra were analyzed in vitro for immunomodulatory activity and an MTT colorimetric assay was conducted to determine the proliferation of mice splenocytes and thymocytes. A bromodeoxyuridine assay was performed to analyze DNA synthesis and the Trypan blue exclusion method was conducted to evaluate the changes in total cell population. The results ...

  3. Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents.

    OpenAIRE

    Eldrige, S R; Butterworth, B E; Goldsworthy, T L

    1993-01-01

    Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohistochemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepatocellula...

  4. Epithelial cell renewal in the digestive gland and stomach of mussels, season, age and tidal regime related variations

    OpenAIRE

    Zaldibar, B.; Cancio, I.; Marigómez, I.

    2008-01-01

    The natural variability in cell proliferation activity in the epithelium of the digestive gland and stomach was investigated in mussels, Mytilus galloprovincialis (Lmk), of different age and tidal level at different seasons. After treating mussels with the thymidine analogue bromodeoxyuridine (BrdU) for 6 hours, BrdU immunohistochemistry was performed every 2 hours for the next 36. The relative proportion of BrdU positive cells was quantified as BrdU labelling (‰). ...

  5. Flow cytometric measurement of RNA synthesis based on bromouridine labelling and combined with measurement of DNA content or cell surface antigen

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J; Larsen, J K

    1993-01-01

    RNA synthesis can be analysed in nuclei or cells labelled with 5-bromouridine (BrUrd) and stained using cross-reacting anti-bromodeoxyuridine (BrdUrd) antibody. Flow cytometric dual parameter analysis of BrUrd incorporation and DNA content in nuclear suspensions of human blood lymphocytes showed ...... synthesis in HL-60 and K-562 cells was measured simultaneous with CD13 expression....

  6. 5-Azacytidine induction of mouse endogenous type C virus and suppression of DNA methylation.

    OpenAIRE

    Niwa, O; Sugahara, T

    1981-01-01

    5-Azacytidine was found to induce the expression of BALB:virus-1 and BALB:virus-2 from K-BALB cells and ecotropic endogenous virus from AKR2B cells. Efficiency of the induction was high and comparable to that by 5-bromodeoxyuridine. The level of methylcytosine in newly synthesized DNA was drastically decreased when K-BALB cells were treated with 5-azacytidine. There was an inverse relationship between the level of DNA modification and the frequency of virus expression.

  7. Effects of vitamin antioxidant supplementation on cell kinetics of patients with adenomatous polyps.

    OpenAIRE

    Cahill, R J; O'Sullivan, K R; Mathias, P M; Beattie, S.; Hamilton, H; O'Morain, C

    1993-01-01

    Colonic crypt cell proliferation is used as an indicator of risk of colorectal carcinoma. Subjects with adenomatous polyps and cancer have an increased cell proliferation and a shift of the proliferative zone towards the apex of the crypt. Epidemiological and in vitro studies have confirmed a link between vitamins A, E, C, beta-carotene, and colorectal cancer. In vitro bromodeoxyuridine immunohistochemical technique was used to assess the effect of daily oral supplementation with vitamin E (1...

  8. Cellular proliferation in the rat pineal gland during postnatal development

    OpenAIRE

    Carvajal, J.C.; Carbajo, S.; Gómez Esteban, M.B.; Alvarez-Morujo Suárez, A.J.; Muñoz Barragan, L.

    1998-01-01

    To establish a possible correlation between the rate of cellular proliferation and already documented functional and morphological characteristics of the rat pineal gland during postnatal development, the bromodeoxyuridine labelling method was used to evaluate the fraction of cells at the S phase of the cell cycle in paraffin sections from I-, 7-, 14- and 28-day-old rats. Numerical density, taken as an indirect measure of cell hypertrophy, was also evaluated. D...

  9. Aggravation of Chronic Stress Effects on Hippocampal Neurogenesis and Spatial Memory in LPA1 Receptor Knockout Mice

    OpenAIRE

    Castilla-Ortega, Estela; Hoyo-Becerra, Carolina; Pedraza, Carmen; Chun, Jerold; Rodríguez de Fonseca, Fernando; Estivill-Torrús, Guillermo; Santín, Luis J.

    2011-01-01

    Background The lysophosphatidic acid LPA1 receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA1 receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory. Methodology/Principal Findings Male LPA1-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous marker...

  10. Excision repair and patch size in UV-irradiated bacteriophage T4.

    OpenAIRE

    Yarosh, D B; Rosenstein, B S; Setlow, R B

    1981-01-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  11. Interactions with the young down-regulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers

    OpenAIRE

    Frédéric Levy

    2014-01-01

    New neurons are continuously added in the dentate gyrus and the olfactory bulb of mammalian brain. While numerous environmental factors controlling survival of newborn neurons have been extensively studied, regulation by social interactions is less documented. We addressed this question by investigating the influence of parturition and interactions with the young on neurogenesis in sheep mothers. Using Bromodeoxyuridine, a marker of cell division, in combination with markers of neuronal matur...

  12. Etanercept Attenuates Traumatic Brain Injury in Rats by Reducing Brain TNF-α Contents and by Stimulating Newly Formed Neurogenesis

    OpenAIRE

    Chong-Un Cheong; Ching-Ping Chang; Chien-Ming Chao; Bor-Chih Cheng; Chung-Zhing Yang; Chung-Ching Chio

    2013-01-01

    It remains unclear whether etanercept penetrates directly into the contused brain and improves the outcomes of TBI by attenuating brain contents of TNF- α and/or stimulating newly formed neurogenesis. Rats that sustained TBI are immediately treated with etanercept. Acute neurological and motor injury is assessed in all rats the day prior to and 7 days after surgery. The numbers of the colocalizations of 5-bromodeoxyuridine and doublecortin specific markers in the contused brain injury that oc...

  13. The fate of Müller’s glia following experimental retinal detachment: nuclear migration, cell division, and subretinal glial scar formation

    OpenAIRE

    Lewis, Geoffrey P.; Chapin, Ethan A.; Luna, Gabriel; Linberg, Kenneth A.; Fisher, Steven K.

    2010-01-01

    Purpose To study the fate of Müller’s glia following experimental retinal detachment, using a “pulse/chase” paradigm of bromodeoxyuridine (BrdU) labeling for the purpose of understanding the role of Müller cell division in subretinal scar formation. Methods Experimental retinal detachments were created in pigmented rabbit eyes, and 3 days later 10 µg of BrdU was injected intravitreally. The retinas were harvested 4 h after the BrdU was administered (i.e., day 3) or on days 4, 7, and 21 post d...

  14. Modulation of ganglioside expression in human melanoma cell lines

    International Nuclear Information System (INIS)

    Cell surface gangliosides in human melanoma cell lines were modulated by pretreatment and adaptation to 6-thioguanine and 5-bromo-deoxyuridine. Chemo- and radiation sensitivities were compared in original cell lines and modulated cells by the human tumor colony-forming assay. Modulated cells showed decreased expression of cell surface GM2 and GD2 gangliosides. This reduction was correlated with increased resistance to bleomycin, vincristine, cisplatin and radiation treatment. These results suggest that cell surface GM2 and GD2 ganglioside expression in human melanoma cells is intimately associated with several cellular biological properties, such as drug or radiation sensitivity and cellular differentiation. (author)

  15. Use of the frequencies of micronuclei as quantitative indicators of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes: comparison of two methods

    International Nuclear Information System (INIS)

    The yield of radiation-induced micronuclei in human lymphocytes was assessed by two methods, i.e., by incorporating bromodeoxyuridine or by inhibiting cytokinesis by cytochalasin for identification of cells which have undergone one cell division. The cytochalasin block method was found to be more efficient with a capacity to detect between 60 and 90% of the induced fragments. Dose-response characteristics and the results of fractionation experiments indicate that the yield of micronuclei reflects both classes of acentric fragments, i.e., those associated and independent of exchange type of aberrations. 8 refs.; 2 figs.; 5 tabs

  16. 3-[3-(3-florophenyl-2-propyn-1-ylthio)-1, 2, 5-thiadiazol-4-yl]-1, 2, 5, 6-tetrahydro-1- methylpyridine oxalate, a novel xanomeline derivative, improves neural cells proliferation and survival in adult mice

    OpenAIRE

    Zhang, Xiaoliang; Gong, Qiang; Zhang, Shuang; Wang, Lin; Hu, Yinghe; Shen, Haiming; Dong, Suzhen

    2012-01-01

    The present study analyzed the influence of 3-[3-(3-florophenyl-2-propyn-1-ylthio)-1, 2, 5-thiadiazol-4-yl]-1, 2, 5, 6-tetrahydro-1-methylpyridine oxalate (EUK1001), a novel xanomeline derivative of the M1/M4 receptor agonist, on hippocampal neurogenesis in adult C57BL6 mice. Results showed that 15-day EUK1001 treatment via intraperitoneal injection promoted neural cell proliferation in the dentate gyrus, although cell differentiation did not change. The majority of bromodeoxyuridine-positive...

  17. Changing bone marrow micro-environment during development of acute myeloid leukaemia in rats

    DEFF Research Database (Denmark)

    Mortensen, B T; Jensen, P O; Helledie, N;

    1998-01-01

    bromodeoxyuridine (BrdUrd) to identify DNA replicating cells. The leukaemia progressed slowly until day 27 after which a rapid deterioration could be observed leading to severe changes over the following 5 d. In whole blood there was evidence of progressing metabolic acidosis. In bone marrow the fraction of......The Brown Norwegian rat transplanted with promyelocytic leukaemic cells (BNML) has been used as a model for human acute myeloid leukaemia. We have previously shown that both the blood supply to the bone marrow and the metabolic rate decrease in relation to the leukaemic development in these rats...

  18. DNA fork displacement rates in human cells

    International Nuclear Information System (INIS)

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 μm/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions. (Auth.)

  19. Thyroid Regeneration: Characterization of Clear Cells After Partial Thyroidectomy

    OpenAIRE

    Ozaki, Takashi; Matsubara, Tsutomu; Seo, Daekwan; Okamoto, Minoru; Nagashima, Kunio; Sasaki, Yoshihito; Hayase, Suguru; Murata, Tsubasa; Liao, Xiao-Hui; Hanson, Jeffrey; Rodriguez-Canales, Jaime; Thorgeirsson, Snorri S.; Kakudo, Kennichi; Refetoff, Samuel; Kimura, Shioko

    2012-01-01

    Although having the capacity to grow in response to a stimulus that perturbs the pituitary-thyroid axis, the thyroid gland is considered not a regenerative organ. In this study, partial thyroidectomy (PTx) was used to produce a condition for thyroid regeneration. In the intact thyroid gland, the central areas of both lobes served as the proliferative centers where microfollicles, and bromodeoxyuridine (BrdU)-positive and/or C cells, were localized. Two weeks after PTx, the number of BrdU-posi...

  20. Inhibitory Effect of Diabetes on Proliferation of Vascular Smooth Muscle After Balloon Injury in Rat Aorta

    OpenAIRE

    Dahlfors, Gunilla; Chen, Yun; Gustafsson, Bertil; Arnqvist, Hans J.

    2000-01-01

    The effect of streptozotocin-induced diabetes on cell proliferation in rat aortic intima-media, as well as on local gene expression of transforming growth factor-β1 (TGF-β1) was studied. TGF-β1 mRNA was measured by solution hybridization and TGF-β1 protein by ELISA. Proliferation was measured by bromodeoxyuridine incorporation into DNA two days after balloon injury. All BrdU-labelled cells observed were smooth muscle cells. After a diabetes duration of 2 and 4 weeks, labelled cells were signi...

  1. Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    Jindou Jiang; Xingyao Bu; Meng Liu; Peixun Cheng

    2012-01-01

    Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury.

  2. Mammalin cochlear supporting cells transdifferentiation into outer hair cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure. Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU) was given from day 16 to day 40 of this recovery phase. Hearing was assessed by overt acoustic behavior and auditory brainstem responses analysis, which was performed one day prior to the first injection and a day after the last injection (day 16). On day 40 animals were sacrificed for detection o...

  3. Exhaustive in vivo labelling of plasmid DNA with BrdU for intracellular detection in non-viral transfection of mammalian cells

    OpenAIRE

    Jérôme, Valérie; Heider, Andreas; Schallon, Anja; Freitag, Ruth

    2009-01-01

    Abstract The study of the non-viral gene delivery process at the molecular level, e.g. during the trans-fection of mammalian cells, is currently limited by difficulties to specifically detect the trans-fected plasmid DNA within the cells. Herein we describe the in vivo production of 5-bromodeoxyuridine- (5-BrdU) labelled plasmid DNA by a thymine-requiring Escherichia coli strain leading to 92 ? 15 % BrdU incorporation while minimizing plasmid structure alteration. The labelled plas...

  4. Cultured mouse embryos metabolize benzo[a]pyrene during early gestation: genetic differences detectable by sister chromatid exchange.

    OpenAIRE

    Galloway, S M; Perry, P E; Meneses, J. (Julio); Nebert, D W; Pedersen, R A

    1980-01-01

    Mouse embryos explanted at 7 1/2 or 8 1/2 days of gestation were cultured in medium containing benzo[a]pyrene and supplemented with 5-bromodeoxyuridine to allow detection of sister chromatid exchanges. The murine Ah locus regulates the inducible metabolism of polycyclic hydrocarbons such as benzo[a]pyrene. A high frequency of sister chromatid exchange was induced by benzo[a]pyrene in embryos from three Ah-"responsive" inbred strains (BALB/cDub, C3H/AnfCum, and C57BL/6N); there was little or n...

  5. Subretinal posterior pole injury induces selective proliferation of RPE cells in the periphery in in vivo studies in pigs

    DEFF Research Database (Denmark)

    Kiilgaard, Jens Folke; Prause, Jan U; Prause, Michala;

    2007-01-01

    transplantation (n= 4), or both (n= 1) in the left eye. RPE cell proliferation was assayed by injection of the thymidine analogue 5-bromodeoxyuridine (5-BrdU) at postoperative day 0 and 1. RPE cells in S-phase were identified by their incorporation of 5-BrdU, as detected by immunohistochemistry. The in vitro...... proliferation of primary RPE isolates from the peripheral and central retina was assayed by a colorimetric assay and by [(3)H]thymidine incorporation. RESULTS: After subretinal surgery, in vivo incorporation of 5-BrdU was seen in peripheral RPE cells in 8 of 10 surgically treated eyes, but never in central RPE...

  6. Phosphoinositide-3-kinases p110alpha and p110beta mediate S phase entry in astroglial cells in the marginal zone of rat neocortex

    Directory of Open Access Journals (Sweden)

    Rabea eMüller

    2013-03-01

    Full Text Available In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine. Only in astroglial cells harvested from the marginal zone of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with bromodeoxyuridine, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110 and p110were essential for S phase entry. p110 diminished the level of the p27Kip1 which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110phosphorylated and inhibited glycogen synthase kinase-3which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

  7. Changes in Expression of Connexin 32, Bile Canaliculus-Like Structures, and Localization of Alkaline Phosphatase in Primary Cultures of Fetal Rat Hepatocytes

    International Nuclear Information System (INIS)

    We devised an experimental design in primary cultures of fetal rat hepatocytes for studying hepatocyte differentiation over a short period. In the present study, hepatocytes were first cultured for 3 days in dexamethasone-supplemented medium and then for an additional 3 days in dexamethasone- or epidermal growth factor-supplemented medium. In hepatocytes cultured continuously in dexamethasone-supplemented medium, the expression of connexin 32 increased and bile canaliculus-like structures and localization of alkaline phosphatase in the plasma membrane around bile canaliculus-like structures were maintained. Few cells incorporated bromodeoxyuridine. On the other hand, in most of the hepatocytes cultured in epidermal growth factor-supplemented medium, the expression of connexin 32 was minimally recognized, bile canaliculus-like structures were shortened or eliminated, and alkaline phosphatase was localized as numerous fine spots throughout the cytoplasm. More than 20% of all hepatocytes incorporated bromodeoxyuridine. The present study suggests that in hepatocytes, there is a close relationship among connexin 32 expression, the maintenance of bile canaliculus-like structures, and the localization of alkaline phosphatase to the plasma membrane around the bile canaliculus-like structures, and this indicates that the present experimental model is useful for studying hepatocyte differentiation over a short period

  8. Chronic intermittent hypoxia alters ventilatory and metabolic responses to acute hypoxia in rats.

    Science.gov (United States)

    Morgan, Barbara J; Adrian, Russell; Wang, Zun-Yi; Bates, Melissa L; Dopp, John M

    2016-05-15

    We determined the effects of chronic exposure to intermittent hypoxia (CIH) on chemoreflex control of ventilation in conscious animals. Adult male Sprague-Dawley rats were exposed to CIH [nadir oxygen saturation (SpO2), 75%; 15 events/h; 10 h/day] or normoxia (NORM) for 21 days. We assessed the following responses to acute, graded hypoxia before and after exposures: ventilation (V̇e, via barometric plethysmography), V̇o2 and V̇co2 (analysis of expired air), heart rate (HR), and SpO2 (pulse oximetry via neck collar). We quantified hypoxia-induced chemoreceptor sensitivity by calculating the stimulus-response relationship between SpO2 and the ventilatory equivalent for V̇co2 (linear regression). An additional aim was to determine whether CIH causes proliferation of carotid body glomus cells (using bromodeoxyuridine). CIH exposure increased the slope of the V̇e/V̇co2/SpO2 relationship and caused hyperventilation in normoxia. Bromodeoxyuridine staining was comparable in CIH and NORM. Thus our CIH paradigm augmented hypoxic chemosensitivity without causing glomus cell proliferation. PMID:26917692

  9. Electrophysiological functional recovery in a rat model of spinal cord hemisection injury following bone marrow-derived mesenchymal stem cell transplantation under hypothermia

    Institute of Scientific and Technical Information of China (English)

    Dong Wang; Jianjun Zhang

    2012-01-01

    Following successful establishment of a rat model of spinal cord hemisection injury by resecting right spinal cord tissues, bone marrow stem cells were transplanted into the spinal cord lesions via the caudal vein while maintaining rectal temperature at 34 ± 0.5°C for 6 hours (mild hypothermia). Hematoxylin-eosin staining showed that astrocytes gathered around the injury site and formed scars at 4 weeks post-transplantation. Compared with rats transplanted with bone marrow stem cells under normal temperature, rats transplanted with bone marrow stem cells under hypothermia showed increased numbers of proliferating cells (bromodeoxyuridine-positive cells), better recovery of somatosensory-evoked and motor-evoked potentials, greater Basso, Beattie, and Bresnahan locomotor rating scores, and an increased degree of angle in the incline plate test. These findings suggested that hypothermia combined with bone marrow mesenchymal stem cells transplantation effectively promoted electrical conduction and nerve functional repair in a rat model of spinal cord hemisection injury.

  10. DNA damage and repair in Stylonychia lemnae (Ciliata, Protozoa)

    International Nuclear Information System (INIS)

    Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-3H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei

  11. Recombinant human erythropoietin increases cerebral cortical width index and neurogenesis following ischemic stroke

    Institute of Scientific and Technical Information of China (English)

    Zhongmin Wen; Peiji Wang

    2012-01-01

    The cerebral cortical expansion index refers to the ratio between left and right cortex width and is recognized as an indicator for cortical hyperplasia. Cerebral ischemia was established in CB-17 mice in the present study, and the mice were subsequently treated with recombinant human erythropoietin via subcutaneous injection. Results demonstrated that cerebral cortical width index significantly increased. Immunofluorescence detection showed that the number of nuclear antigen antibody/5-bromodeoxyuridine-positive cells at the infarction edge significantly increased. Correlation analysis revealed a negative correlation between neurological scores and cortical width indices in rats following ischemic stroke. These experimental findings suggested that recombinant human erythropoietin promoted cerebral cortical hyperplasia, increased cortical neurogenesis, and enhanced functional recovery following ischemic stroke.

  12. Spontaneous Proliferation in Organotypic Cultures of Mouse Cochleae

    Institute of Scientific and Technical Information of China (English)

    DING Da-lian; WANG Jian; YU Zhi-ping; JIANG Hai-yan; WANG Ping; Richard Salvi

    2008-01-01

    Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative polymerase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.

  13. Inhibition by blueberries (bilberries) and extract from milk thistle of rat forestomach hyperplasia induced by oral smokeless tobacco (Swedish snus).

    Science.gov (United States)

    Nilsson, Robert; Mićić, Mileva; Filipović, Jelena; Šobot, Ana Valenta; Drakulić, Dunja; Stanojlović, Miloš; Joksiċ, Gordana

    2016-04-01

    The aim of this study was to identify palatable additives which have a significant protective action against soft tissue changes in the oral cavity caused by Swedish smokeless tobacco ("snus"), and that satisfy existing legal requirements. Although the cancer risk from snus is extremely low, long term use may result in highly undesirable keratotic lesions and associated epithelial abnormalities in the oral cavity. The rat forestomach, which is vulnerable to the irritative action of non-genotoxic compounds like butylated hydroxyanisole, propionic acid as well as snus, was chosen as an experimental model. Studied toxicological endpoints included histopathology and cellular proliferation based on DNA incorporation of bromodeoxyuridine. After 6 weeks' exposure, blueberries (bilberries) and an extract from the common milk thistle were found to exert a highly significant inhibition of cell proliferation induced by snus in the rat forestomach epithelium, indicating a potential protection with respect soft tissue changes in the human oral cavity. PMID:26828024

  14. Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

    DEFF Research Database (Denmark)

    Jensen, Pia; Gramsbergen, Jan-Bert; Zimmer, Jens; Widmer, Hans R; Meyer, Morten

    2011-01-01

    Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of...... rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in......, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH...

  15. Sister chromatid exchanges

    International Nuclear Information System (INIS)

    Sister chromatid exchanges (SCEs) are cytological manifestations of DNA double-strand breakage and rejoining at homologous sites between the two chromatids of a chromosome. The occurrence of SCEs was deduced from the transformation of small ring chromosomes to large ring chromosomes following cell division. Using tritiated thymidine as marker and microautoradiography for detection, others demonstrated the occurrence of SCEs from the silver grain pattern on the sister chromatids. This method was eventually replaced by cytochemical methods. One showed that if cells were grown in medium containing 5-bromo-deoxyuridine (BrdUrd) for two cycles, the sister chromatids can be distinguished by the differential quenching of the fluorescence of the fluorochrome Hoechst 33258. Reduced staining with Giemsa stain of the BrdUrd-incorporated chromatids was also found to be useful in differentiating sister chromatids. The authors review in this chapter only those studies which have implications on the origin of SCEs

  16. Intra-arterial infusion of radiosensitizer (BUdR) combined with hypofractionated irradiation and chemotherapy for primary treatment of osteogenic sarcoma

    International Nuclear Information System (INIS)

    Combined modality treatment was given in nine patients of osteogenic sarcoma wherein the tumor was unresectable because of location or amputation was refused. This alternative to massive surgery comprised hypofractionated irradiation, intra-arterial infusion of the radiosensitizer 5'-bromodeoxyuridine (BUdR) and adjuvant systemic chemotherapy. Local control was achieved in seven of the nine patients. Four survived, all without evidence of disease at 6, 7.1, 8.8, and 10.5 years after completion of irradiation. Pulmonary metastases developed in six patients - of whom one survives, following high-dose pulmonary irradiation and additional chemotherapy. Significant soft-tissue injury occurred in five patients. On the basis of our experience, the authors believe that new approaches using modifications of external beam irradiation with different fractionation schedules or better radiosensitizing compounds may hold promise for patients with non-resectable osteosarcoma

  17. Estrogen and progesterone stimulate Schwann cell proliferation in a sex- and age-dependent manner

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1999-01-01

    The effects of estrogen and progesterone on Schwann cell proliferation were studied in cultured segments of the rat sciatic nerve from adult male, female, and newborn rats, by measurement of [3H thymidine incorporation or bromo-deoxy-uridine- (BrdU)-labelling and immunocytochemistry. Estrogen (100...... Schwann cells from male rats at high concentrations. The proliferative effects of estrogen and progesterone were blocked when the segments were cultured in the presence of inhibitors of their respective receptors, ICI 128 780 and zk 112994. The data suggest that Schwann cells possess distinct receptors...... for estrogen and progesterone and that these receptors may be involved in the control of Schwann cell proliferation. It also shows that the response of Schwann cells to sex hormones varies with sex and perhaps also with age....

  18. The effect of suppressor of cytokine signaling 3 on GH signaling in beta-cells

    DEFF Research Database (Denmark)

    Rønn, Sif G; Hansen, Johnny A; Lindberg, Karen;

    2002-01-01

    . Furthermore, using Northern blot analysis it was shown that SOCS-3 can completely inhibit GH-induced insulin production in these cells. Finally, 5-bromodeoxyuridine incorporation followed by fluorescence-activated cell sorting analysis showed that SOCS-3 inhibits GH-induced proliferation of INS-1 cells. These......GH is an important regulator of cell growth and metabolism. In the pancreas, GH stimulates mitogenesis as well as insulin production in beta-cells. The cellular effects of GH are exerted mainly through activation of the Janus kinase-signal transducer and activator of transcription (STAT) pathway....... Recently it has been found that suppressors of cytokine signaling (SOCS) proteins are able to inhibit GH-induced signal transduction. In the present study, the role of SOCS-3 in GH signaling was investigated in the pancreatic beta-cell lines RIN-5AH and INS-1 by means of inducible expression systems. Via...

  19. Chromosome condensation and segmentation

    International Nuclear Information System (INIS)

    Some aspects of chromosome condensation in mammalians -humans especially- were studied by means of cytogenetic techniques of chromosome banding. Two further approaches were adopted: a study of normal condensation as early as prophase, and an analysis of chromosome segmentation induced by physical (temperature and γ-rays) or chemical agents (base analogues, antibiotics, ...) in order to show out the factors liable to affect condensation. Here 'segmentation' means an abnormal chromosome condensation appearing systematically and being reproducible. The study of normal condensation was made possible by the development of a technique based on cell synchronization by thymidine and giving prophasic and prometaphasic cells. Besides, the possibility of inducing R-banding segmentations on these cells by BrdU (5-bromodeoxyuridine) allowed a much finer analysis of karyotypes. Another technique was developed using 5-ACR (5-azacytidine), it allowed to induce a segmentation similar to the one obtained using BrdU and identify heterochromatic areas rich in G-C bases pairs

  20. Test of radiation damage enhancement due to incorporation of BrUdR into DNA using chromatid aberrations

    International Nuclear Information System (INIS)

    Monte Carlo track structure calculations, leading to an estimation of the magnitude of enhancement of radiation damage due to the incorporation of the halogenated pyrimidine, bromodeoxyuridine (BrUdR) a thymine analog, into DNA have been made. The increase in the yield of double strand breaks for various degrees of substitution in one (monofilarly) or both strands (bifilarly) have been calculated. To test these calculations, quantitative selected radiation-induced aberrations have been obtained in Chinese hamster (V79) fibroblast chromosomes having various patterns of BrUdR substitution following irradiation with 250 kV X rays. Free ''breaks'' and achromatic lesions ''gaps'' show no appreciable sensitizations, but breaks involved in chromatid interchanges show significant enhancement though of lower magnitude than theoretical predictions

  1. Excision repair in ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome, and Bloom's syndrome after treatment with ultraviolet radiation and N-acetoxy-2-acetylaminofluorene

    International Nuclear Information System (INIS)

    Excision repair of damage due to ultraviolet radiation, N-acetoxy-2-acetylaminofluorene and a combination of both agents was studied in normal human fibroblasts and various cells from cancer prone patients (ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome and Bloom's syndrome). Three methods giving similar results were used: unscheduled DNA synthesis by radioautography, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and loss of sites sensitive to an ultraviolet endonuclease. All cell lines were proficient in repair of ultraviolet and acetoxy acetylaminofluorene damage and at saturation doses of both agents repair was additive. We interpret these data as indicating that the rate limiting step in excision repair of ultraviolet and acetoxy acetylaminofluorene is different and that there are different enzyme(s) working on incision of both types of damages. (Auth.)

  2. Radiosensitivity of chromosomes in two successive mitotic cycles of human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Luchnik, N.V.; Poryadkova, N.A.

    1988-11-01

    A culture of human lymphocytes was irradiated with /gamma/-quanta in a dose of 0.5 Gy with different ratios of cells in first (M1) and second (M2) mitotic cycle and the frequency of aberrations induced at stage G2 was analyzed. With increase in interval of time between the start of culturing and irradiation, total yield of aberrations increased in a regular way. However, if the M1:M2 ratio is considered, then it turns out that in M2 chromosomes are /approximately/1.5 times more sensitive than in M1: within the limits of each cycle, radiosensitivity is constant and does not depend on its duration. It was established in accordance with data of other authors that 5-bromodeoxyuridine (5-BdU) increases radiosensitivity materially.

  3. In vivo measurement of DNA synthesis rates of colon epithelial cells in carcinogenesis

    International Nuclear Information System (INIS)

    We describe here a highly sensitive technique for measuring DNA synthesis rates of colon epithelial cells in vivo. Male SD rats were given 2H2O (heavy water). Colon epithelial cells were isolated, DNA was extracted, hydrolyzed to deoxyribonucleosides, and the deuterium enrichment of the deoxyribose moiety was determined by gas chromatographic/mass spectrometry. Turnover time of colon crypts and the time for migration of cells from basal to top fraction of the crypts were measured. These data were consistent with cell cycle analysis and bromodeoxyuridine labeling. By giving different concentrations of a promoter, dose-dependent increases in DNA synthesis rates were detected, demonstrating the sensitivity of the method. Administration of a carcinogen increased DNA synthesis rates cell proliferation in all fractions of the crypt. In conclusion, DNA synthesis rates of colon epithelial cells can be measured directly in vivo using stable-isotope labeling. Potential applications in humans include use as a biomarker for cancer chemoprevention studies

  4. 8-hydroxy-dipropylaminotetralin promotes neural plasticity in epileptic rats with depression

    Institute of Scientific and Technical Information of China (English)

    Ping Yang; Meizhen Sun; Liang Li; Yihua Shen

    2012-01-01

    Rats with chronic pilocarpine-induced temporal lobe epilepsy complicated with depression were studied. Anti-5-bromodeoxyuridine immunofluorescence staining and Timms staining showed that neurogenesis within the hippocampal dentate gyrus and mossy fiber sprouting were increased in model rats. Neurogenesis within the hippocampal dentate gyrus was further enhanced, while mossy fiber sprouting was decreased in model rats administered carbamazepine alone or in combination with the 5-hydroxytryptamine 1A receptor agonist, 8-hydroxy-dipropylaminotetralin (0.1 and 1 mg/kg). Among the groups, the effect was the most significant in rats receiving carbamazepine in conjunction with 1 mg/kg 8-hydroxy-dipropylaminotetralin. Thus, high dose 8-hydroxy-dipropylaminotetralin can improve neural plasticity in epileptic rats with depression.

  5. Cell kinetic study on the relation between irradiation hypogeusia and taste buds in rats

    Energy Technology Data Exchange (ETDEWEB)

    Kubota, Hideharu; Furumoto, Keiichi [Nippon Dental Univ., Tokyo (Japan)

    1998-12-01

    The present study was designed to elucidate the mechanism of hypogeusia caused by irradiation. X-ray treatment at 10 Gy or 20 Gy was given to the maxillofacial region including the tongue in rats, and the involvement of taste bud for hypogeusia was investigated. In addition, cytological kinetics were immunohistologically studied using bromodeoxyuridine in the taste bud and in the lingual mucosal epithelium. The following results were obtained: In the 10 Gy group, the number of taste bud become less after the exposure, but no hypogeusia was observed during the experimental period. In the 20 Gy group, any labeled taste bud was not observed on the 7th day, and all taste buds disappeared by the 10th day. In the lingual mucosal epithelium, the number of basal cells decreased to the minimum, and the body weight and total water intake decreased coincidently in the 20 Gy group, which were few in the 10 Gy group. (author)

  6. In situ cell cycle kinetics in bone marrow biopsies following sequential infusions of IUdR/BrdU in patients with hematopoietic malignancies.

    Science.gov (United States)

    Raza, A; Yousuf, N; Bohkari, S A; Sheikh, Y; Akhtar, S; Chughtai, S; Umerani, A; Mehdi, S A; Miller, M A; Masterson, M

    1992-01-01

    Examination of the proliferative characteristics of myeloblasts was undertaken in situ in bone marrow (BM) biopsies of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) following sequential infusions of iodo- (IUdR) and bromodeoxyuridine (BrdU). The ability to identify S-phase cells which have incorporated both or either one of the labels in vivo by using two monoclonal antibodies in vitro permitted the measurement of labeling index (LI) and durations of S-phase (Ts) and the total cell cycle (Tc) both from the BM aspirates and biopsies. While the LI is 2-3 times higher in biopsies, Ts and Tc are fairly comparable in the two samples in 8/10 cases (p = 0.02 and 0.003 respectively). Advantages associated with the determination of cell cycle parameters in BM biopsies have been discussed at length. PMID:1560677

  7. Osteoclast nuclei of myeloma patients show chromosome translocations specific for the myeloma cell clone: a new type of cancer-host partnership?

    DEFF Research Database (Denmark)

    Levin Andersen, Thomas; Boissy, Patrice; Sondergaard, T E;

    2007-01-01

    study demonstrates that bone-resorbing osteoclasts from myeloma patients contain nuclei with translocated chromosomes of myeloma B-cell clone origin, in addition to nuclei without these translocations, by using combined FISH and immunohistochemistry on bone sections. These nuclei of malignant origin are...... proximity of myeloma cells. Similar hybrid cells were generated in myeloma cell-osteoclast co-cultures, as revealed by tracing myeloma nuclei using translocations, bromo-deoxyuridine, or the Y chromosome of male myeloma cells in female osteoclasts. These observations indicate that hybrid cells can originate......A major clinical manifestation of bone cancers is bone destruction. It is widely accepted that this destruction is not caused by the malignant cells themselves, but by osteoclasts, multinucleated cells of monocytic origin that are considered to be the only cells able to degrade bone. The present...

  8. Deficient leukemia inhibitory factor signaling in muscle precursor cells from patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Broholm, Christa; Brandt, Claus; Schultz, Ninna S; Nielsen, Anders R; Pedersen, Bente K; Scheele, Camilla

    2012-01-01

    The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response...... to LIF. The mRNA and protein expressions of LIF and its receptor (LIFR) were measured in skeletal muscle biopsies from healthy individuals and patients with type 2 diabetes by use of qPCR and Western blot. LIF signaling and response were studied following administration of recombinant LIF and si......RNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts...

  9. Use of halogenated thymidine analogs as clinical radiosensitizers: rationale, current status, and future prospects: non-hypoxic cell sensitizers

    International Nuclear Information System (INIS)

    The halogenated pyrimidine analogs, bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) have been recognized as potential clinical radiosensitizers for over two decades. In vivo and in vitro experimental studies document that radiosensitization is directly dependent on the amount of thymidine replacement in DNA by these analogs. Based on recent in vivo and clinical pharmacology studies on continuous intravenous infusions of these drugs, clinical trials are underway evaluating the potential of radiosensitization in high grade gliomass and other poorly radioresponsive tumors using the technically safer intravenous route of administration. In this paper, the authors review the basic strategy for the use of these analogs, the ongoing clinical trials and the potential areas for future experimental and clinical studies

  10. The insulin-like growth factors I and II stimulate proliferation of different types of Schwann cells

    DEFF Research Database (Denmark)

    Sondell, M; Svenningsen, Åsa Fex; Kanje, M

    1997-01-01

    A combination of immunocytochemistry for glial specific antigens and bromodeoxyuridine (BrdU) and teasing was used to identify proliferating cells in cultured rat sciatic nerve segments. The nerve segments were exposed to insulin, or the insulin-like growth factors IGF-I and IGF-II. Teasing in...... combination with BrdU immunocytochemistry showed that around 93% of the proliferating cells in the nerve segments were Schwann cells. Immunostaining for BrdU and GFAP (glial fibrillary acid protein) showed that IGF-II enhanced proliferation of Schwann cells surrounding unmyelinated nerve fibres. In contrast......, truncated IGF-I promoted proliferation of Schwann cells of myelinated nerve fibres while insulin increased proliferation of both cell types....

  11. Effect of post-treatments with caffeine during G2 on the frequencies of chromosome-type aberrations produced by X-rays in human lymphocytes during G0 and G1

    International Nuclear Information System (INIS)

    Human lymphocytes were irradiated with X-rays in G0 and G1, grown in the presence of 5-bromodeoxyuridine, and harvested at different times from 48 to 80 h after stimulation. Some cultures were exposed to 2.5-5 mM caffeine during the last 3 h before harvesting. The frequencies of chromosome-type aberrations were scored in first division (M1) metaphases. The post-treatment with caffeine increased the frequencies of mitoses and chromosome-type aberrations in irradiated cultures. The results suggest that cells carrying chromosome-type aberrations are delayed in G2 and that caffeine increases the frequencies of aberrations in dividing cells by removing this G2-block. (author)

  12. Exogenous glucagon-like peptide-2 (GLP-2) augments GLP-2 receptor mRNA and maintains proglucagon mRNA levels in resected rats

    DEFF Research Database (Denmark)

    Koopmann, Matthew C; Nelson, David W; Murali, Sangita G;

    2008-01-01

    BACKGROUND: Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent proglucagon-derived hormone that stimulates intestinal adaptive growth. Our aim was to determine whether exogenous GLP-2 increases resection-induced adaptation without diminishing endogenous proglucagon and GLP-2 receptor...... expression. METHODS: Rats underwent transection or 70% jejunoileal resection +/- GLP-2 infusion (100 microg/kg body weight/d) and were fed a semipurified diet with continuous infusion of GLP-2 or saline by means of jugular catheter. After 7 days, body weight, mucosal cellularity (dry mass, protein and DNA......), crypt-villus height, and crypt cell proliferation (by bromodeoxyuridine staining) were determined. Plasma bioactive GLP-2 (by radioimmunoassay), proglucagon and GLP-2 receptor mRNA expression (by Northern blot and real-time reverse transcriptase quantitative polymerase chain reaction) were measured. GLP...

  13. HAIR CELL-LIKE CELL GENERATION INDUCED BY NATURE CULTURE OF ADULT RAT AUDITORY EPITHELIUM

    Institute of Scientific and Technical Information of China (English)

    Liu Hui; Zhu Hongliang; Li Shengli; Yao Xiaobao; Wang Xiaoxia

    2006-01-01

    Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.

  14. Effect of lymphocytes culture variations on the mitotic index and on the dicentric yield following gamma radiation exposure

    International Nuclear Information System (INIS)

    Fundamentals of biological dosimetry are described in the International Atomic Energy Agency manual, but all over the world each laboratory is using its own protocol. To test the influence of protocol variations, some blood samples were exposed to 0.5 Gy of gamma radiation and mitotic index and dicentric rates were measured under different experimental conditions. The effect of seven parameters [bromodeoxyuridine (BrdU), phytohaemagglutinin and colcemide concentrations, blood and medium volumes, culture duration and incubation temperature] was tested using a Placket and Burman experimental design. The analysis reveals that the mitotic index was influenced by the concentration of BrdU, medium and blood volumes, the culture duration and the temperature. However, none of the factors has a significant impact on the yield of dicentrics. The dicentric assay is robust against reagent variations within the range tested. These results could be used by relevant laboratories as elements of their procedures robustness in any event requiring such demonstration. (authors)

  15. The PPARα Agonist Fenofibrate Preserves Hippocampal Neurogenesis and Inhibits Microglial Activation After Whole-Brain Irradiation

    International Nuclear Information System (INIS)

    Purpose: Whole-brain irradiation (WBI) leads to cognitive impairment months to years after radiation. Numerous studies suggest that decreased hippocampal neurogenesis and microglial activation are involved in the pathogenesis of WBI-induced brain injury. The goal of this study was to investigate whether administration of the peroxisomal proliferator-activated receptor (PPAR) α agonist fenofibrate would prevent the detrimental effect of WBI on hippocampal neurogenesis. Methods and Materials: For this study, 129S1/SvImJ wild-type and PPARα knockout mice that were fed either regular or 0.2% wt/wt fenofibrate-containing chow received either sham irradiation or WBI (10-Gy single dose of 137Cs γ-rays). Mice were injected intraperitoneally with bromodeoxyuridine to label the surviving cells at 1 month after WBI, and the newborn neurons were counted at 2 months after WBI by use of bromodeoxyuridine/neuronal nuclei double immunofluorescence. Proliferation in the subgranular zone and microglial activation were measured at 1 week and 2 months after WBI by use of Ki-67 and CD68 immunohistochemistry, respectively. Results: Whole-brain irradiation led to a significant decrease in the number of newborn hippocampal neurons 2 months after it was performed. Fenofibrate prevented this decrease by promoting the survival of newborn cells in the dentate gyrus. In addition, fenofibrate treatment was associated with decreased microglial activation in the dentate gyrus after WBI. The neuroprotective effects of fenofibrate were abolished in the knockout mice, indicating a PPARα-dependent mechanism or mechanisms. Conclusions: These data highlight a novel role for PPARα ligands in improving neurogenesis after WBI and offer the promise of improving the quality of life for brain cancer patients receiving radiotherapy.

  16. Cellular heredity in haploid cultures of somatic cells. Comprehensive report, April 1975--June 1977

    International Nuclear Information System (INIS)

    This report reviews genetic studies carried out since 1975 on a haploid cultured cell line from frog embryos (ICR 2A). Although a single chromosome set would be expected to facilitate recovery of recessive mutants, experiments suggested that cell culture variants might arise through processes more complex than the selection of simple mutational changes. Therefore, the objectives of the work reported here have been to throw light on just how cell culture variants arise in this system. First, we have continued to characterize the ICR 2A line, with emphasis on stability of karyotype and DNA content. Second, we have studied in detail the origin of two classes of drug-resistant variants. Bromodeoxyuridine resistance of the thymidine deficiency type has been shown to arise through sequential loss of two forms of thymidine-phosphorylating enzyme; loss of the second form of enzyme is complex, suggesting that changes more complex than simple recessive mutations may be involved. Another form of resistance, in which tolerance of high levels of bromodeoxyuridine is found in cells that continue to express thymidine kinase, remains under study. Variants resistant to microtubule inhibitors were isolated. It was found that these haploid strains have properties distinguishing them from analogous resistant strains isolated from diploid mammalian cell cultures in other laboratories. In order to understand better how mutagens are involved in the origin of cell culture variants, we have examined the effect of different forms of DNA repair on the frequency of drug-resistant colonies induced by ultraviolet radiation. Preliminary experiments suggest that the frequency of such colonies is greater when repair takes place through (presumably error-prone) dark repair than when (error-free) photoreversal is allowed to occur. Such experiments can determine whether new phenotypes arise from alterations in DNA, and thus whether, in a broad sense, they are likely to be mutational in nature

  17. Peptide hormone exendin-4 stimulates subventricular zone neurogenesis in the adult rodent brain and induces recovery in an animal model of Parkinson's disease.

    Science.gov (United States)

    Bertilsson, Göran; Patrone, Cesare; Zachrisson, Olof; Andersson, Annica; Dannaeus, Karin; Heidrich, Jessica; Kortesmaa, Jarkko; Mercer, Alex; Nielsen, Elisabet; Rönnholm, Harriet; Wikström, Lilian

    2008-02-01

    We investigated the effects of exendin-4 on neural stem/progenitor cells in the subventricular zone of the adult rodent brain and its functional effects in an animal model of Parkinson's disease. Our results showed expression of GLP-1 receptor mRNA or protein in the subventricular zone and cultured neural stem/progenitor cells isolated from this region. In vitro, exendin-4 increased the number of neural stem/progenitor cells, and the number of cells expressing the neuronal markers microtubule-associated protein 2, beta-III-tubulin, and neuron-specific enolase. When exendin-4 was given intraperitoneally to naïve rodents together with bromodeoxyuridine, a marker for DNA synthesis, both the number of bromodeoxyuridine-positive cells and the number of neuronal precursor cells expressing doublecortin were increased. Exendin-4 was tested in the 6-hydroxydopamine model of Parkinson's disease to investigate its possible functional effects in an animal model with neuronal loss. After unilateral lesion and a 5-week stabilization period, the rats were treated for 3 weeks with exendin-4. We found a reduction of amphetamine-induced rotations in animals receiving exendin-4 that persisted for several weeks after drug administration had been terminated. Histological analysis showed that exendin-4 significantly increased the number of both tyrosine hydroxylase- and vesicular monoamine transporter 2-positive neurons in the substantia nigra. In conclusion, our results show that exendin-4 is able to promote adult neurogenesis in vitro and in vivo, normalize dopamine imbalance, and increase the number of cells positive for markers of dopaminergic neurons in the substantia nigra in a model of Parkinson's disease. PMID:17803225

  18. Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique.

    Science.gov (United States)

    Bhaskara, Srividya

    2016-01-01

    Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples. PMID:26863264

  19. Cellular heredity in haploid cultures of somatic cells. Comprehensive report, April 1975--June 1977. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Freed, J.J.

    1977-07-01

    This report reviews genetic studies carried out since 1975 on a haploid cultured cell line from frog embryos (ICR 2A). Although a single chromosome set would be expected to facilitate recovery of recessive mutants, experiments suggested that cell culture variants might arise through processes more complex than the selection of simple mutational changes. Therefore, the objectives of the work reported here have been to throw light on just how cell culture variants arise in this system. First, we have continued to characterize the ICR 2A line, with emphasis on stability of karyotype and DNA content. Second, we have studied in detail the origin of two classes of drug-resistant variants. Bromodeoxyuridine resistance of the thymidine deficiency type has been shown to arise through sequential loss of two forms of thymidine-phosphorylating enzyme; loss of the second form of enzyme is complex, suggesting that changes more complex than simple recessive mutations may be involved. Another form of resistance, in which tolerance of high levels of bromodeoxyuridine is found in cells that continue to express thymidine kinase, remains under study. Variants resistant to microtubule inhibitors were isolated. It was found that these haploid strains have properties distinguishing them from analogous resistant strains isolated from diploid mammalian cell cultures in other laboratories. In order to understand better how mutagens are involved in the origin of cell culture variants, we have examined the effect of different forms of DNA repair on the frequency of drug-resistant colonies induced by ultraviolet radiation. Preliminary experiments suggest that the frequency of such colonies is greater when repair takes place through (presumably error-prone) dark repair than when (error-free) photoreversal is allowed to occur. Such experiments can determine whether new phenotypes arise from alterations in DNA, and thus whether, in a broad sense, they are likely to be mutational in nature.

  20. Nuclear vasohibin-2 promotes cell proliferation by inducing G0/G1 to S phase progression.

    Science.gov (United States)

    Ge, Qianqian; Zhou, Jia; Tu, Min; Xue, Xiaofeng; Li, Zhanjun; Lu, Zipeng; Wei, Jishu; Song, Guoxin; Chen, Jianmin; Guo, Feng; Jiang, Kuirong; Miao, Yi; Gao, Wentao

    2015-09-01

    As a member of the vasohibin (VASH2) family, VASH2 is localized intracellularly as a nuclear and cytoplasmic type. Cytoplasmic VASH2 is associated with carcinoma angiogenesis and malignant transformation and promotes cancer growth. However, the function of nuclear VASH2 has yet to be investigated. The aim of the present study was to detect the nuclear VASH2 expression profile in human organs and tissues by protein microarray technique. To examine the function of nuclear VASH2, we analyzed the relationship between nuclear VASH2 and Ki-67, and stably constructed VASH2 overexpression and knockdown in LO2 and HepG2 cell lines, based on a previous study in hepatic cells. The study was conducted using bromodeoxyuridine, immunofluorescent staining, western blot analysis and flow cytometry. Nuclear VASH2 was highly expressed in actively dividing cells in normal and cancer tissues. There was a significant positive correlation between nuclear VASH2 and Ki-67, indicating that nuclear VASH2 positively correlated with cell proliferation in normal and cancer tissues. The bromodeoxyuridine (BrdU) proliferation test showed that nuclear VASH2 increased the S-phase population and promoted cell proliferation, while VASH2 knockdown reduced BrdU absorbance. Cell cycle analysis revealed that nuclear VASH2 overexpression increased the S-phase population in LO2 and HepG2 cells, while nuclear VASH2 knockdown reduced the S-phase population and increased the G0/G1 population. The findings of this study challenge the classic view of VASH2, which was previously reported as an angiogenesis factor. Furthermore, to the best of our knowledge, these results are the first clinical data indicating that nuclear VASH2, but not cytoplasmic VASH2, promotes cell proliferation by driving the cell cycle from the G0/G1 to S phase. PMID:26177649

  1. Glucagonlike Peptide 2 Analogue Teduglutide

    Science.gov (United States)

    Chaturvedi, Lakshmi S.; Basson, Marc D.

    2015-01-01

    IMPORTANCE Short bowel syndrome occurs when a shortened intestine cannot absorb sufficient nutrients or fluids. Teduglutide is a recombinant analogue of human glucagonlike peptide 2 that reduces dependence on parenteral nutrition in patients with short bowel syndrome by promoting enterocytic proliferation, increasing the absorptive surface area. However, enterocyte function depends not only on the number of cells that are present but also on differentiated features that facilitate nutrient absorption and digestion. OBJECTIVE To test the hypothesis that teduglutide impairs human intestinal epithelial differentiation. DESIGN AND SETTING We investigated the effects of teduglutide in the modulation of proliferation and differentiation in human Caco-2 intestinal epithelial cells at a basic science laboratory. This was an in vitro study using Caco-2 cells, a human-derived intestinal epithelial cell line commonly used to model enterocytic biology. EXPOSURE Cells were exposed to teduglutide or vehicle control. MAINOUTCOMESAND MEASURES We analyzed the cell cycle by bromodeoxyuridine incorporation or propidium iodide staining and flow cytometry and measured cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. We used quantitative reverse transcription–polymerase chain reaction to assay the expression of the enterocytic differentiation markers villin, sucrase-isomaltase, glucose transporter 2 (GLUT2), and dipeptidyl peptidase 4 (DPP-4), as well as that of the putative differentiation signals schlafen 12 (SLFN12) and caudal-related homeobox intestine-specific transcription factor (Cdx2). Villin promoter activity was measured by a luciferase-based assay. RESULTS The MTS assay demonstrated that teduglutide increased cell numbers by a mean (SD) of 10% (2%) over untreated controls at a maximal 500nM (n = 6, P < .05). Teduglutide increased bromodeoxyuridine-positive cells vs untreated controls by a mean (SD

  2. Combination of single walled carbon nanotubes/graphene oxide with paclitaxel: a reactive oxygen species mediated synergism for treatment of lung cancer

    Science.gov (United States)

    Arya, Neha; Arora, Aditya; Vasu, K. S.; Sood, A. K.; Katti, Dhirendra S.

    2013-03-01

    Heterogeneity in tumors has led to the development of combination therapies that enable enhanced cell death. Previously explored combination therapies mostly involved the use of bioactive molecules. In this work, we explored a non-conventional strategy of using carbon nanostructures (CNs) [single walled carbon nanotube (SWNT) and graphene oxide (GO)] for potentiating the efficacy of a bioactive molecule [paclitaxel (Tx)] for the treatment of lung cancer. The results demonstrated enhanced cell death following combination treatment of SWNT/GO and Tx indicating a synergistic effect. In addition, synergism was abrogated in the presence of an anti-oxidant, N-acetyl cysteine (NAC), and was therefore shown to be reactive oxygen species (ROS) dependent. It was further demonstrated using bromodeoxyuridine (BrdU) incorporation assay that treatment with CNs was associated with enhanced mitogen associated protein kinase (MAPK) activation that was ROS mediated. Hence, these results for the first time demonstrated the potential of SWNT/GO as co-therapeutic agents with Tx for the treatment of lung cancer.Heterogeneity in tumors has led to the development of combination therapies that enable enhanced cell death. Previously explored combination therapies mostly involved the use of bioactive molecules. In this work, we explored a non-conventional strategy of using carbon nanostructures (CNs) [single walled carbon nanotube (SWNT) and graphene oxide (GO)] for potentiating the efficacy of a bioactive molecule [paclitaxel (Tx)] for the treatment of lung cancer. The results demonstrated enhanced cell death following combination treatment of SWNT/GO and Tx indicating a synergistic effect. In addition, synergism was abrogated in the presence of an anti-oxidant, N-acetyl cysteine (NAC), and was therefore shown to be reactive oxygen species (ROS) dependent. It was further demonstrated using bromodeoxyuridine (BrdU) incorporation assay that treatment with CNs was associated with enhanced

  3. Defective and enhanced postreplication repair in classical and variant xeroderma pigmentosum cells treated with N-acetoxy-2-acetylaminofluorene

    International Nuclear Information System (INIS)

    Xeroderma pigmentosum (XP) cells proficient in the excision repair of pyrimidine dimers (XP variants) were also found to be proficient in the excision repair of N-2-acetoxyacetylaminofluorene (AAAF)-induced lesions in their DNA, as assayed by the photolysis of 5-bromodeoxyuridine incorporated during repair. However, the time in which the small segments of newly synthesized DNA, made immediately after treatment of cells with AAAF, were joined together to form DNA of parental size by a process called postreplication repair was long in the XP variant and classical cells. Although increasing doses of AAAF increased the time for making daughter DNA of parental size for variant and classical XP cells, AAAF did not appear to affect this process in normal human cells. Treatment of variant and classical XP cells with a relatively small dose (2.5 μM) of AAAF or 2.5 J/sq m of uv radiation several hr before a 2- to 3-fold-larger dose decreased the time for the pulse-labeled DNA to appear as parental size

  4. Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [3H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg2+-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

  5. Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

    International Nuclear Information System (INIS)

    Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, the authors conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1). Continuous post-UV treatment with 1mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating of the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists untill the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency. (author). 21 refs.; 4 figs.; 4 tabs

  6. (64)Cu-ATSM therapy targets regions with activated DNA repair and enrichment of CD133(+) cells in an HT-29 tumor model: Sensitization with a nucleic acid antimetabolite.

    Science.gov (United States)

    Yoshii, Yukie; Furukawa, Takako; Matsumoto, Hiroki; Yoshimoto, Mitsuyoshi; Kiyono, Yasushi; Zhang, Ming-Rong; Fujibayashi, Yasuhisa; Saga, Tsuneo

    2016-06-28

    (64)Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a potential theranostic agent targeting the over-reduced state under hypoxia within tumors. Recent clinical Cu-ATSM positron emission tomography studies have revealed a correlation between uptake and poor prognosis; however, the reason is unclear. Here, using a human colon carcinoma HT-29 model, we demonstrated that the intratumoral (64)Cu-ATSM high-uptake regions exhibited malignant characteristics, such as upregulated DNA repair and elevated %CD133(+) cancer stem-like cells. Based on this evidence, we developed a strategy to enhance the efficacy of (64)Cu-ATSM internal radiotherapy (IRT) by inhibiting DNA repair with a nucleic acid (NA) antimetabolite. The results of the analyses showed upregulation of pathways related to DNA repair along with NA incorporation (bromodeoxyuridine uptake) and elevation of %CD133(+) cells in (64)Cu-ATSM high-uptake regions. In an in vivo(64)Cu-ATSM treatment study, co-administration of an NA antimetabolite and (64)Cu-ATSM synergistically inhibited tumor growth, with little toxicity, and effectively reduced %CD133(+) cells. (64)Cu-ATSM therapy targeted malignant tumor regions with activated DNA repair and high concentrations of CD133(+) cells in the HT-29 model. NA antimetabolite co-administration can be an effective approach to enhance the therapeutic effect of (64)Cu-ATSM IRT. PMID:26996296

  7. Inhalation of tobacco smoke induces increased proliferation of urinary bladder epithelium and endothelium in female C57BL/6 mice

    International Nuclear Information System (INIS)

    Cigarette smoking is the major environmental risk factor for bladder cancer in humans. Aromatic amines, potent DNA-reactive bladder carcinogens present in cigarette smoke, contribute significantly. However, increased cell proliferation, caused by direct mitogenesis or in response to cytotoxicity, may also play a role since urothelial hyperplasia has been observed in human cigarette smokers. We examined the urothelial effects of cigarette smoke (whole body inhalation exposure (Teague) system) in female C57BL/6 mice at various times in two studies, including reversibility evaluations. In both studies, no urothelial hyperplasia was observed by light microscopy in any group. However, in study 1, the Ki-67 labeling index (LI) of the urothelium was significantly increased in the smoke exposed group compared to controls through 3 months, but was not present at 6, 9 or 12 months even with continued exposures. In the groups that discontinued smoke exposure, it returned to the same levels as controls or lower. In study 2, the bromodeoxyuridine LI was similar to controls on day 1 but significantly increased at 5 days in the smoke exposed group. In the group that discontinued smoke exposure for 2 days, the LI was increased compared to controls but not significantly. Superficial urothelial cell cytotoxicity and necrosis were detectable by scanning electron microscopy at 5 days. Changes in LI of submucosal endothelial cells generally followed those of the urothelium and effects were reversible upon cessation of exposure. The increased urothelial proliferation appeared to be due to superficial cell cytotoxicity with consequent regeneration

  8. Effect of chlorophyllin on induction of exchanges in sister chromatids by gamma irradiation in mice spermatogonia in vivo

    International Nuclear Information System (INIS)

    Mouse were exposed to different doses of gamma radiation and the effect on Sister Chromatid Exchange (SCE) frequency in spermatogonias was evaluated. The effect was analyzed before and after Bromodeoxyuridine (BrdU) incorporation to determine the interference of such agent with the cellular response induced by radiation. The capacity of chlorophyllin (sodium and Copper salt derivative from chlorophyll) to reduce SCE induction by radiation in normal and BrdU radio sensitized spermatogonia was also determined. The results indicate that there was a significant increase in SCE frequency by gamma radiation exposure in these cells, such effect was higher irradiating after BrdU incorporation than before. This fact confirms previous observations that BrdU sensitizes some cells to SCE induction. With regard to the chlorophyllin effect, it was determined that this salt acts as a radioprotector reducing gamma-rays induced SCE before or after BrdU incorporation Total protection was obtained with 200 μg of chlorophyllin per g of body weight in both protocols. Under the experimental conditions this study there was no evidence of genotoxicity induced by chlorophyllin itself. The results suggest that this agent may act as a radioprotector by scavenging free radicals produced by gamma-radiation which cause DNA lesions that are involved in SCE formation. (Author)

  9. Angiogenic Potency of Bone Marrow Stromal Cells Improved by ex Vivo Hypoxia Prestimulation

    Institute of Scientific and Technical Information of China (English)

    毛晓波; 曾秋棠; 王祥; 曹林生; 白智峰

    2004-01-01

    To study the angiogenic potency of hypoxia-prestimulated bone marrow stromal cells (BMSCs) when transplanted into acute myocardial infarction models of rats. BMSCs were cultured under hypoxia condition for 24 h. Their expression of VEGF was investigated. The rat acute myocardial infarction models were made by coronary artery ligation and divided into 3 groups at random.In normoxia group, twice-passaged BMSCs were labeled with Bromodeoxyuridine (BrdU) and then implanted into the infarction regions and ischemic border of the recipients in 4 weeks. The rats in hypoxia group were implanted with hypoxia-prestimulated BMSCs. In control group, the model rats received only DMEM medium injection. Six-weeks after AMI, the infarction regions were examined to identify the angiogenesis and the expression of the VEGF. Our results showed that viable cells labeled with BrdU could be identified in the host hearts. The infarction regions in normoxia and hypoxia groups had a greater capillary density and increased VEGF expression than the regions in control group. The capillary density and VEGF expression in hypoxia group were higher than in normoxia group. It is concluded that the enhanced expression of VEGF in BMSCs could be induced by ex vivo hypoxia stimulation. BMSCs implantation promoted the angiogenesis in myocardial infarction tissue via supplying exogenic VEGF. Angiogenic potency of bone marrow stromal cells was improved by ex vivo hypoxia prestimulation though the enhanced VEGF expression.

  10. Evidence that cyclophosphamide can to induce exchanges in the sister chromatids (ICH) through secondary injuries; Evidencia de que la ciclofosfamida puede inducir intercambios en las cromatidas hermanas (ICH) a traves de lesiones secundarias

    Energy Technology Data Exchange (ETDEWEB)

    Morales R, P.; Rodriguez R, R. [Instituto Nacional de Investigaciones nucleares, A.P. 18-1027, 11801 Mexico D.F. (Mexico)

    1997-07-01

    By means of the use of destination protocol of ICH inductive injuries (DLI-ICH), it was studied if interchanges in the sister chromatids (ICH) induced by cyclophosphamide (CP), in the second post-treatment division (ICH-2) are produced by secondary injuries or by fresh injuries. For discard between these possibilities it was administered CP at different periods before of the first post-treatment division, taking as reference the administered time for high dose of bromodeoxyuridine (BrdU ) which was approximately at the beginning of this division. The ICH frequencies that occur in the first, the second and the third synthesis stages (S) were determined. It was observed that when the administered CP was four hours before BrdU , the ICH frequencies of the second and the third S were reduced. The frequency of the first ICH increased lightly in relation to those of the normal protocol (0.5 h before BrdU ) and that the supplying of CP six hours before caused almost a total reduction of ICH of second and third S and an important increment of ICH of first S.This was interpreted as evidence that the ICH-2 are product of secondary injuries. (Author)

  11. Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

    Energy Technology Data Exchange (ETDEWEB)

    Jansson, Janet; Edlund, Anna; Hardeman, Fredrik; Jansson, Janet K.; Sjoling, Sara

    2008-05-15

    Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179 mV, -64 mV and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labeled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labeled DNA and DNA from reverse transcription PCR (rt-PCR) showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

  12. Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells

    Energy Technology Data Exchange (ETDEWEB)

    Ayusawa, D.; Koyama, H.; Shimizu, K.; Kaneda, S.; Takeishi, K.; Seno, T.

    1986-10-01

    Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.

  13. Marker evaluation of human breast and bladder cancers

    Energy Technology Data Exchange (ETDEWEB)

    Mayall, B.H.; Carroll, P.R.; Chen, Ling-Chun; Cohen, M.B.; Goodson, W.H. III; Smith, H.S.; Waldman, F.M. (California Univ., San Francisco, CA (USA))

    1990-11-02

    We are investigating multiple markers in human breast and bladder cancers. Our aim is to identify markers that are clinically relevant and that contribute to our understanding of the disease process in individual patients. Good markers accurately assess the malignant potential of a cancer in an individual patient. Thus, they help identify those cancers that will recur, and they may be used to predict more accurately time to recurrence, response to treatment, and overall prognosis. Therapy and patient management may then be optimized to the individual patient. Relevant markers reflect the underlying pathobiology of individual tumors. As a tissue undergoes transformation from benign to malignant, the cells lose their differentiated phenotype. As a generalization, the more the cellular phenotype, cellular proliferation and cellular genotype depart from normal, the more advanced is the tumor in its biological evolution and the more likely it is that the patient has a poor prognosis. We use three studies to illustrate our investigation of potential tumor markers. Breast cancers are labeled in vivo with 5-bromodeoxyuridine (BrdUrd) to give a direct measure of the tumor labeling index. Bladder cancers are analyzed immunocytochemically using an antibody against proliferation. Finally, the techniques of molecular genetics are used to detect allelic loss in breast cancers. 6 refs., 3 figs.

  14. Development of custom-built bone scaffolds using mesenchymal stem cells and apatite-wollastonite glass-ceramics.

    Science.gov (United States)

    Dyson, Jennifer A; Genever, Paul G; Dalgarno, Kenneth W; Wood, David J

    2007-12-01

    There is a clinical need for new bone replacement materials that combine long implant life with complete integration and appropriate mechanical properties. We have used human mesenchymal stem cells (MSCs) to populate porous apatite-wollastonite (A-W) glass-ceramic scaffolds produced by the layer manufacturing technique, selective laser sintering, to create custom-built bone replacements. Confocal and scanning electron microscopy were used to determine optimal seeding densities and to demonstrate that MSCs adhered and retained viability on the surface of A-W scaffolds over a culture period of 21 days. We found a significant increase in the number of MSCs growing on the scaffolds over 7 days. Using bromodeoxyuridine incorporation we demonstrated that MSCs proliferated on the scaffolds. Using real-time PCR we analyzed the expression of the osteogenic markers alkaline phosphatase, collagen type-I, Cbfa-1, osteocalcin, osteonectin, and osteopontin by MSCs cultured in the absence of osteogenic supplements. The expression of the osteogenic markers by MSCs was equivalent to or significantly greater on A-W scaffolds than on tissue culture plastic. We also identified significantly higher alkaline phosphatase activity on A-W compared to a commercial calcium phosphate scaffold. These results indicate for the first time the biocompatibility and osteo-supportive capacity of A-W scaffolds and their potential as patient-specific bone replacement materials. PMID:17764401

  15. 3-[3-(3-florophenyl-2-propyn-1-ylthio)-1, 2, 5-thiadiazol-4-yl]-1, 2, 5, 6-tetrahydro-1- methylpyridine oxalate, a novel xanomeline derivative, improves neural cells proliferation and survival in adult mice

    Institute of Scientific and Technical Information of China (English)

    Xiaoliang Zhang; Qiang Gong; Shuang Zhang; Lin Wang; Yinghe Hu; Haiming Shen; Suzhen Dong

    2012-01-01

    The present study analyzed the influence of 3-[3-(3-florophenyl-2-propyn-1-ylthio)-1, 2, 5-thiadiazol-4-yl]-1, 2, 5, 6-tetrahydro-1-methylpyridine oxalate (EUK1001), a novel xanomeline derivative of the M1/M4 receptor agonist, on hippocampal neurogenesis in adult C57BL6 mice. Results showed that 15-day EUK1001 treatment via intraperitoneal injection promoted neural cell proliferation in the dentate gyrus, although cell differentiation did not change. The majority of bromodeoxyuridine-positive cells co-expressed the immature neuronal marker doublecortin. In addition, the level of neurogenesis in the subventricular zone was not altered. Brain-derived neurotrophic factor mRNA expression was up-regulated following EUK1001 treatment, but no change was observed in expression of camp-responsive element binding protein 1, paired box gene 6, vascular endothelial growth factor alpha, neurogenic differentiation factor 1, and wingless-related mouse mammary tumor virus integration site 3A mRNA. These experimental findings indicated that EUK1001 enhanced proliferation and survival of hippocampal cells, possibly by increasing brain-derived neurotrophic factor expression.

  16. Effect of voluntary running on adult hippocampal neurogenesis in cholinergic lesioned mice

    Directory of Open Access Journals (Sweden)

    Dawe Gavin S

    2009-06-01

    Full Text Available Abstract Background Cholinergic neuronal dysfunction of the basal forebrain is observed in patients with Alzheimer's disease and dementia, and has been linked to decreased neurogenesis in the hippocampus, a region involved in learning and memory. Running is a robust inducer of adult hippocampal neurogenesis. This study aims to address the effect of running on hippocampal neurogenesis in lesioned mice, where septohippocampal cholinergic neurones have been selectively eliminated in the medial septum and diagonal band of Broca of the basal forebrain by infusion of mu-p75-saporin immunotoxin. Results Running increased the number of newborn cells in the dentate gyrus of the hippocampus in cholinergic denervated mice compared to non-lesioned mice 24 hours after injection of bromodeoxyuridine (BrdU. Although similar levels of surviving cells were present in cholinergic depleted animals and their respective controls four weeks after injection of BrdU, the majority of progenitors that proliferate in response to the initial period of running were not able to survive beyond one month without cholinergic input. Despite this, the running-induced increase in the number of surviving neurones was not affected by cholinergic depletion. Conclusion The lesion paradigm used here models aspects of the cholinergic deficits associated with Alzheimer's Disease and aging. We showed that running still increased the number of newborn cells in the adult hippocampal dentate gyrus in this model of neurodegenerative disease.

  17. Mammalin cochlear supporting cells transdifferentiation into outer hair cells

    Institute of Scientific and Technical Information of China (English)

    Liu Siwei; Zhang Shaoqiang; Zhu Hongliang; Li Baiya; Zheng Qingyin; Li Shengli

    2008-01-01

    Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure.Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU)was given from day 16 to day 40 of this recovery phase. Hearing was assessed by overt acoustic behavior and auditory brainstem responses analysis, which was performed one day prior to the first injection and a day after the last injection (day 16). On day 40 animals were sacrificed for detection of cells that could take up BrdU. Results After 15 days of gentamicin treatment, all of the animals were proved to be deafened with significant increases of ABR thresholds,compared with control group. The findings in immunocytochemical stained samples and scanning electron microscopy revealed that BrdU labeled nuclei were observed in the cochlea in all of the deafened animals most commonly in the regions of the first-row and second-row Deiter's cells (DCs) and occasionally in the regions of the third-ruw DCs.Conclusion We suggest that, under sufficient drug and enough time, the bat cochlear supporting cells can directly transdifferentiate into the outer hair cells after aminoglycoside exposure. This transdifferentation process is essential for repair of outer hair cells and recovery of normal function after gentamicin exposure.

  18. A sensitization effect of hematoporphyrin oligomer (HpO) and caffeine for X-ray radiation of skin cancer

    International Nuclear Information System (INIS)

    Human malignant melanoma cells (G-361) were irradiated with X-ray for different irradiation times. The amount of DNA damage was much increased by the treatment of the cells with hematoporphyrin oligomer (HpO) before X-ray irradiation. Furthermore, it was found that the caffeine inhibited effectively the repair of the damaged DNA. Furthermore, HpO was administered before X-ray irradiation of transplantable skin tumor in C3H mice and/or caffeine was to be given the mouse after X-ray irradiation. The growth of the irradiated tumor combined with HpO and/or caffeine was more inhibited, and the necrotic area was more expanded than that of the tumor irradiated with X-ray only. Labeling Index utilizing bromodeoxyuridine (BrdU) in the viable area of the treated tumor decreased when HpO and/or caffeine were administered. The amount of DNA damage more increased in the irradiated tumor cells given HpO than the only irradiated cells. And, the repair of the damaged DNA was more delayed in the irradiated tumor cells combined with HpO and caffeine than those with other treatments. In conclusion, it was considered that HpO might be a radioactive sensitizer and caffeine enhanced the inhibition of the repair of DNA damaged. We hope that the treatment of X-ray radiation combined with HpO and caffeine is useful for clinical treatment of human malignant tumor. (author) 54 refs

  19. Environmental Enrichment Attenuated Sevoflurane-Induced Neurotoxicity through the PPAR-γ Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yupeng Zhao

    2015-01-01

    Full Text Available Sevoflurane is the most widely used inhaled anesthetic. Environmental enrichment (EE can reverse sevoflurane-induced learning and memory impairment in young mice. However, the mechanism by which EE elicits this effect is unclear. The peroxisome proliferator-activated receptor (PPAR regulatory pathway plays a critical role in the regulation of inflammation in central nervous system diseases. In this study, we investigated whether EE attenuates sevoflurane-induced learning and memory disability via the PPAR signaling pathway. Six-day-old mice were treated with 3% sevoflurane for 2 hours daily from postnatal day 6 (P6 to P8. Then, the mice were treated with EE. The effects of sevoflurane on learning and memory function, PPAR-γ expression in the brain, and the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and 5-bromodeoxyuridine-positive cells in the hippocampus were determined. Sevoflurane induced neuronal apoptosis and neurogenesis inhibition, which may impair learning and memory in young mice. Furthermore, sevoflurane downregulated PPAR-γ expression. Both EE and the PPAR-γ agonist, rosiglitazone, attenuated sevoflurane-induced neuronal apoptosis, neurogenesis inhibition, and learning and memory impairment. Our findings suggest that EE ameliorated sevoflurane-induced neurotoxicity and learning and memory impairment through the PPAR-γ signaling pathway. PPAR-γ may be a potential therapeutic target for preventing or treating sevoflurane-induced neurotoxicity.

  20. The functional organization of mitochondrial genomes in human cells

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    Kimura Hiroshi

    2004-05-01

    Full Text Available Abstract Background We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24 containing mitochondria tagged with the yellow fluorescent protein. Results Mitochondrial DNA is organized in ~475 discrete foci containing 6–10 genomes. These foci (nucleoids are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 × 10-3 μm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time ~43 min long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins – that is, a ribosomal protein (S6, a nascent peptide associated protein (NAC, and the translocase in the outer membrane (Tom22. Conclusions The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.

  1. Modification of radiation response in mammalian cells: the repair and fixation of x-ray induced potentially lethal damage

    International Nuclear Information System (INIS)

    The effects of anisotonic salt treatments, polyamines, dimethyl sulfoxide (DMSO), 5'-bromodeoxyuridine (BrdUrd) and hyperthermia on the repair and fixation of X-ray-induced radiation damage were examined in V79 Chinese hamster lung fibroblasts, Chinese hamster ovary cells, normal and ataxia telangiectasis (AT) human cells. Significant repair of potentially lethal damage (PLD) was observed when cells were held in Earle's balanced salt solutions, or in plateau phase, following exposure to X-radiation; a much greater recovery was observed when cells were incubated at 37 degrees C between irradiation and subsequent exposure to anisotonic salts. Cells exposed to 1 or 2 mol/L DMSO or 10-5 mol/L BrdUrd exhibited PLD repair patterns comparable to control cells. Contrary to the results of others, human AT homozygotes, like normal human cells, did sustain radiation damage that could be fixed or repaired by anisotonic salt treatment. The extent of damage fixation was dose-dependent and was observed at doses as low as 0.25 Gy. A greater degree of PLD was fixed when cells were irradiated at 0 degrees C rather than 37 degrees C, suggesting that repair occurs during irradiation. While heat damage was not susceptible to fixation by anisotonic salt treatment, the synergistic enhancement of X-ray damage due to heat was fixed in a pattern dependent on the heating temperature

  2. Measurement of DNA repair in Chinese hamster fibroblasts employing flow cytometry and monoclonal antibodies to DNA adducts

    International Nuclear Information System (INIS)

    The authors examined the utility of measuring DNA repair in single cells employing flow cytometric quantitation of fluorescent monoclonal antibodies directed against specific DNA adducts. Two antibodies were employed; the first directed against single strand (ss) bromodeoxyuridine (anti-BrdUrd) and the second against UV light induced ss-thymine dimers (anti-TT). Sensitivity with both monoclonals was highly dependent on DNA denaturation, with the most effective shown to be a 0.5N HCl histone extraction followed by 50% formamide for 30 min at 800C. Unscheduled synthesis following 30 J/m/sup 2/ UV irradiation in Gl/GO plateau phase CHO cells was demonstrated employing the anti-BrdUrd AB method combined with DNA counter-staining with propidium iodide. Data suggest that anti-BrdUrd Ab recognition of newly replicated sequences following UV irradiation may be strongly dependent on chromatin conformation. A linear correlation was observed for mean anti-TT AB fluorescence and UV dose up to a total of 3000 J/m/sup 2/. Also, a rapid reduction in cellular fluorescence, presumably reflecting dimer excision was observed when the cells were returned to 370C before fixation. Finally, data from various repair deficient CHO cells will be compared employing these methods

  3. Relative biological effectiveness of carbon ions for causing fatal liver failure after partial hepatectomy in mice

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    Tomizawa, Minoru; Miyamoto, Tadaaki; Kato, Hirotoshi; Otsu, Hiroshi [National Inst. of Radiological Sciences, Chiba (Japan)

    2000-06-01

    To evaluate the acute phase damage to liver by carbon ions, BALB/c mice were irradiated with carbon ions or X-rays after two-thirds partial hepatectomy, and their survival was followed. The 50% lethal dose within 60 days (LD{sub 50/60}) was 42.2{+-}0.25 Gy (standard error) for X-rays, and 22.7{+-}0.25 Gy for carbon ions. The relative biological effectiveness (RBE) of carbon ions was 1.86 (95% confident limits: 1.69-2.04) as calculated from the LD{sub 50/60}. Mice irradiated at much higher doses, 60 Gy of X-rays or 24 Gy of carbon ions, showed significantly higher serum ammonia levels and lower serum albumin levels than normal, suggesting hepatic failure as a cause of death. Hepatocytes showed karyorrhexis and karyolysis in carbon ion irradiated and spotty necrosis in X-ray irradiated mice, suggesting nuclear damage. Mice irradiated with LD{sub 50} of X-rays or carbon ions had a remarkably lower bromodeoxyuridine (BrdU) labeling index and mitotic index than control. Treatments with both BrdU and vincristine showed that none of the hepatocytes that synthesized DNA after irradiation completed mitosis, indicating G2 arrest. The liver weight of irradiated mice significantly decreased depending on the dose. Carbon ions as well as X-rays damaged hepatocytes directly and suppressed liver regeneration leading to fatal liver failure. (author)

  4. Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin

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    M Vaccarezza

    2009-03-01

    Full Text Available Although several studies have shown that the serum levels of osteoprotegerin (OPG are significantly elevated in patients affected with atherosclerotic lesions in coronary and peripheral arteries, the cellular source and the role of OPG in the physiopathology of atherosclerosis are not completely defined. Therefore, we aimed to investigate the potential contribution of mesenchymal stem cells in the production/release of OPG. OPG was detectable by immunohistochemistry in aortic and coronary atherosclerotic plaques, within or in proximity of intimal vascular smooth muscle cells (SMC. In addition, bone marrow mesenchymal stem cell (MSC-derived vascular SMC as well as primary aortic SMC released in the culture supernatant significantly higher levels of OPG with respect to MSCderived endothelial cells (EC or primary aortic EC. On the other hand, in vitro exposure to full-length human recombinant OPG significantly increased the proliferation rate of aortic SMC cultures, as monitored by bromodeoxyuridine incorporation. Taken together, these data suggest that OPG acts as an autocrine/paracrine growth factor for vascular SMC, which might contribute to the progression of atherosclerotic lesions.

  5. Effect of delta opioid receptor activation on spatial cognition and neurogenesis in cerebral ischemic rats.

    Science.gov (United States)

    Wang, Shu-Yan; Duan, Ya-Le; Zhao, Bing; Wang, Xiang-Rui; Zhao, Zheng; Zhang, Guang-Ming

    2016-05-01

    This study aimed to investigate whether a selective delta opioid receptor agonist, [D-Ala2, D-Leu5]-Enkephalin (DADLE), regulates neurogenesis in the hippocampus of ischemic rats. Using an intracerebral cannula, rats were subjected to cerebral ischemia using the standard four-vessel occlusion. DADLE (2.5nmol), DADLE (2.5nmol) with naltrindole (NAL) (2.5nmol), or vehicle was administered at the onset of reperfusion. Bromodeoxyuridine (BrdU, 100mg/kg, intraperitoneal) was used to label newly formed cells from days 1 to 7 after ischemia. Immunohistochemistry was used to evaluate cell proliferation and apoptosis and differentiation 7days 28 days, respectively, after ischemia. Morris water maze test was conducted to test spatial learning and memory 23-27 days after ischemia. We found that DADLE treatment improved performance in the Morris water maze test, promoted proliferation and differentiation of newly formed neurons, and inhibited differentiation into astrocytes in a rat model of cerebral ischemia. Furthermore, the protective effects of DADLE were significantly reversed by co-administration of NAL (Pstrategy for cerebral ischemia. PMID:27016387

  6. In vivo and in vitro protein-DNA interactions at the distal oestrogen response element of the chicken vitellogenin gene: evidence for the same protein binding to this sequence in hen and rooster liver.

    Science.gov (United States)

    McEwan, I J; Saluz, H P; Jost, J P

    1991-03-01

    The major egg white protein, vitellogenin, is synthesized in a tissue specific and oestradiol dependent manner in the liver of egg-laying hens. In this paper, we describe a detailed study of the protein-DNA interactions at the distal oestrogen response element (ERED) located 600 bp upstream of the start of transcription. In vivo footprinting of hepatocytes from adult hens and roosters with 0.5-0.0005% dimethylsulphate (DMS) revealed, at critical concentrations of DMS, protection of distinct guanosine residues within the ERED and adjacent downstream sequence in both cases. From this, it was concluded that there were proteins present in both tissues binding to this region in vivo. In vitro studies using missing base contact probing and proteolytic clipping band shift assays with hen and rooster liver nuclear extracts identified the ERE binding protein to be the same or very closely related in both tissues. Furthermore, the protein from rooster nuclear extracts bound to the ERE sequence even when the DNA was methylated at CpG dinucleotides, u.v. cross-linking experiments performed with bromodeoxyuridine substituted ERE, revealed that a nuclear protein with Mr of about 75,000-80,000 bound specifically to this sequence. These studies demonstrate that apart from the oestrogen receptor, at least one other protein can interact specifically with the chicken vitellogenin ERE, independently of hormonal expression of the gene. PMID:2009219

  7. Polysaccharopeptide enhances the anticancer activity of doxorubicin and etoposide on human breast cancer cells ZR-75-30.

    Science.gov (United States)

    Wan, Jennifer Man-Fan; Sit, Wai-Hung; Louie, Jimmy Chun-Yu

    2008-03-01

    In search of natural bioactive microbial compounds with adjuvant properties, we have previously showed that the polysaccharopeptide (PSP), isolated from Chinese medicinal mushroom Coriolus versicolor, was able to enhance the cytotoxicity of certain S-phase targeted-drugs on human leukemic HL-60 cells via some cell-cycle and apoptotic-dependent pathways. The present study aimed to investigate whether the synergism of mechanisms of PSP with certain chemotherapeutic drugs also applies to human breast cancer. PSP treatment enhanced the cytotoxicity of doxorubicin (Doxo), etoposide (VP-16) but not cytarabine (Ara-C). Bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometry analysis estimated a longer DNA synthesis time (Ts) for the PSP treated cancerous cells suggesting that PSP enhanced the apoptotic effect of Doxo and VP-16 via creating an S-phase trap in the human breast cancer cell line ZR-75-30. The participation of PSP in the apoptotic machinery of the chemotherapeutic agents was further supported by a reduced ratio of protein expression of Bcl-xL/Bax of the cancer cells. This study provides further insight into the synergistic mechanisms of PSP and supports the hypothesis that the anticancer potentials of PSP is not limited to leukemia but may also be used as an adjuvant therapy for breast cancers. PMID:18292947

  8. Reduced hippocampal dentate cell proliferation and impaired spatial memory performance in aged epileptic rats

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    Clarissa F Cavarsan

    2013-07-01

    Full Text Available Increased adult neurogenesis is observed after training in hippocampal-dependent tasks and also after acutely induced status epilepticus (SE although the specific roles of these cells are still a matter of debate. In this study, we investigated hippocampal cell proliferation and differentiation and the spatial learning performance in young or aged chronically epileptic rats. Status was induced by pilocarpine in 3 or 20-month old rats. Either two or twenty months later, rats were treated with bromodeoxyuridine (BrdU and subsequently underwent to 8-day schedule of water maze tests. As expected, learning curves were faster in young than in aged animals (P<0.001. Chronically epileptic animals exhibited impaired learning curves compared to age-matched controls. Interestingly, the duration of epilepsy (2 or 20 months did not correlate with the memory impairment of aged epileptic animals. The number of BrdU-positive cells was greater in young epileptic subjects than in age-matched controls. In contrast, cell proliferation was not increased in aged epileptic animals, irrespective of the time of SE induction. Finally, dentate cell proliferation was not related to performance in the water maze. Based on the present results we conclude that even though aging and epilepsy lead to impairments in spatial learning, their effects are not additive.

  9. Cyclin D1 expression during rat mammary tumor development and its potential role in the resistance of the Copenhagen rat

    International Nuclear Information System (INIS)

    Resistance to mammary tumorigenesis in Copenhagen rats is associated with loss of early preneoplastic lesions known as intraductal proliferations. The cause of this disappearance, however, is unknown. There were no differences in the numbers of lesions in mammary whole-mounts prepared from Copenhagen or Wistar-Furth rats at 20 or 30 days after N-methyl-N-nitrosourea treatment, but at 37 days there were significantly fewer lesions in Copenhagen glands. Furthermore, lesions in Copenhagen glands were exclusively intraductal proliferations, whereas in Wistar-Furth glands more advanced lesions were also present. Immunohistochemical staining showed frequent cyclin D1 overexpression in Wistar-Furth lesions at 37 days, but not in Copenhagen lesions. There were, however, no differences in p16INK4a protein expression, bromodeoxyuridine labeling and apoptotic indices, or mast cell infiltration between Copenhagen and Wistar-Furth lesions at any time. Overexpression of cyclin D1 in preneoplastic lesions may be important in the development of mammary tumors in susceptible rats, although this overexpression does not appear to cause significant changes in cell kinetics. Furthermore, the low levels of cyclin D1 expression in Copenhagen intraductal proliferations may play a role in the resistance of these rats to mammary tumorigenesis

  10. Increased adult hippocampal neurogenesis is not necessary for wheel running to abolish conditioned place preference for cocaine in mice.

    Science.gov (United States)

    Mustroph, M L; Merritt, J R; Holloway, A L; Pinardo, H; Miller, D S; Kilby, C N; Bucko, P; Wyer, A; Rhodes, J S

    2015-01-01

    Recent evidence suggests that wheel running can abolish conditioned place preference (CPP) for cocaine in mice. Running significantly increases the number of new neurons in the hippocampus, and new neurons have been hypothesised to enhance plasticity and behavioral flexibility. Therefore, we tested the hypothesis that increased neurogenesis was necessary for exercise to abolish cocaine CPP. Male nestin-thymidine kinase transgenic mice were conditioned with cocaine, and then housed with or without running wheels for 32 days. Half of the mice were fed chow containing valganciclovir to induce apoptosis in newly divided neurons, and the other half were fed standard chow. For the first 10 days, mice received daily injections of bromodeoxyuridine (BrdU) to label dividing cells. On the last 4 days, mice were tested for CPP, and then euthanized for measurement of adult hippocampal neurogenesis by counting the number of BrdU-positive neurons in the dentate gyrus. Levels of running were similar in mice fed valganciclovir-containing chow and normal chow. Valganciclovir significantly reduced the numbers of neurons (BrdU-positive/NeuN-positive) in the dentate gyrus of both sedentary mice and runner mice. Valganciclovir-fed runner mice showed similar levels of neurogenesis as sedentary, normal-fed controls. However, valganciclovir-fed runner mice showed the same abolishment of CPP as runner mice with intact neurogenesis. The results demonstrate that elevated adult hippocampal neurogenesis resulting from running is not necessary for running to abolish cocaine CPP in mice. PMID:25393660

  11. Synchrotron infrared spectromicroscopy as a novel bioanalytical microprobe for individual living cells: Cytotoxicity considerations

    Energy Technology Data Exchange (ETDEWEB)

    Holman, Hoi-Ying N.; Bjornstad, Kathleen A.; McNamara, Morgan P.; Martin, Michael C.; McKinney, Wayne R.; Blakely, Eleanor A.

    2001-12-12

    Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy is a newly emerging analytical tool capable of monitoring the biochemistry within an individual living mammalian cell in real time. This unique technique provides infrared (IR)spectra, hence chemical information, with high signal-to-noise at spatial resolutions as fine as 3 to 10 microns. Mid-IR photons are too low in energy (0.05-0.5 eV) to either break bonds or to cause ionization, and the synchrotron IR beam has been shown to produce minimal sample heating. However, an important question remains, ''Does the intense synchrotron beam induce any cytotoxic effects in living cells?'' In this work, we present the results from a series of standard biological assays to evaluate any short-and/or long-term effects on cells exposed to the synchrotron radiation-based infrared (SR-IR) beam. Cell viability was tested using alcian blue dye-exclusion and colony formation assays. Cell-cycle progression was tested with bromodeoxyuridine (BrdU) uptake during DNA synthesis. Cell metabolism was tested using an 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. All control, 5-, 10-, and 20-minute SR-IR exposure tests (267 total and over 1000 controls) show no evidence of cytotoxic effects. Concurrent infrared spectra obtained with each experiment confirm no detectable chemistry changes between control and exposed cells.

  12. 12-O-Tetradecanoyl-phorbol-13-acetate and its relationship to SCE induction in Syrian and Chinese hamster cells

    International Nuclear Information System (INIS)

    12-O-Tetradecanoyl-phorbol-13-acetate (TPA) in conditions that produce enhancement of ultraviolet light (UV) and x-irradiation Syrian hamster embryo cell (HEC) transformation did not cause further increase in the sister chromatid exchange (SCE) frequency induced by UV and x-irradiation, two physical carcinogens that differ in their mode of DNA interaction and efficiency of SCE induction. Several factors which might influence SCE induction by TPA were studied on HEC and Chinese hamster V79-4 cells. Heat-inactivated serum was used because of the possibility that a serum component may interfere with TPA ability to cause SCE. TPA effect on SCE was determined at the first and second division post treatment on cells exposed to different 5-bromodeoxyuridine (BrdUrd) concentrations. Independent of BrdUrd concentration (1-10μg/ml medium) and the number of cells divisions post treatment, TPA (0.01-2μg/ml medium) was ineffective in inducing SCE in exponentially and stationary HEC cultures cultivated in medium supplemented with heat-inactivated serum. Also, TPA did not increase the SCE frequency in V79-4 Chinese hamster cells cultured in heat-activated or noninactivated serum. Although SCE induction, a cellular response to carcinogen-induced DNA damage, may be important for the induction of transformation by environmental agents, the enhancement of transformation frequency caused by TPA occurs without further DNA alterations involved in SCE formation

  13. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

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    Jian Wang

    2016-01-01

    Full Text Available After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF, Adipose-derived Stem Cells (ASCs and those labeled by superparamagnetic iron oxide (SPIO nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT assay, proliferation by cell counting and bromodeoxyuridine (BrdU incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF, Insulin-like Growth Factor-1 (IGF-1, Transforming Growth Factor Beta 1 (TGF-β1, genetic markers comprising Stem Cell Antigen-1 (Sca1, Octamer-4 (Oct-4, ATP-binding Cassette Subfamily B Member 1 (ABCB1, adipogenic marker genes containing Lipoprotein Lipase (LPL, Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ, and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1 and Osterix (OSX. Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.

  14. β-Asarone Reverses Chronic Unpredictable Mild Stress-Induced Depression-Like Behavior and Promotes Hippocampal Neurogenesis in Rats

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    Haiying Dong

    2014-04-01

    Full Text Available In this study, we investigated the influence of β-asarone, the major ingredient of Acorus tatarinowii Schott, on depressive-like behavior induced by the chronic unpredictable mild stresses (CUMS paradigm and to clarify the underlying mechanisms. The results show that β-asarone treatment partially reversed the CUMS-induced depression-like behaviors in both the forced swim and sucrose preference tests. The behavioral effects were associated with increased hippocampal neurogenesis indicated by bromodeoxyuridine (BrdU immunoreactivity. β-Asarone treatment significantly increased the expression of brain-derived neurotrophic factor (BDNF at levels of transcription and translation. Moreover, CUMS caused significant reduction in ERK1/2 and CREB phosphorylation, both of which were partially attenuated by β-asarone administration. It is important to note that β-asarone treatment had no effect on total levels or phosphorylation state of any of the proteins examined in ERK1/2-CREB pathway in no stress rats, suggesting that β-asarone acts in a stress-dependent manner to block ERK1/2-CREB signaling. We did not observe a complete reversal of depression-like behaviors to control levels by β-asarone. To our knowledge, the present study is the first to demonstrate that adult neurogenesis is involved in the antidepressant-like behavioral effects of β-asarone, suggesting that β-asarone is a promising candidate for the treatment of depression.

  15. Isolation and preliminary characterization of u.v.-sensitive mutants from the human cell line EUE

    International Nuclear Information System (INIS)

    Five u.v. light-sensitive clones were isolated in the EUE cell line by means of a modified form of the original 5-bromodeoxyuridine (BUdR)-light method worked out by Puck and Kao for the isolation of nutritional mutants. A cell population was mutagenized with ethylmethanesulfonate. After the expression time, cells were u.v.-irradiated and incubated with BUdR to label excision patches in repair proficient cells. A subsequent irradiation with black light caused DNA strand breakage in BUdR-substituted cells. During BUdR treatment, hydroxyurea and a fluorochrome (Hoechst 33258) were added to possibly enhance the analogue incorporation into DNA and to increase the photolability of BUdR containing sequences, respectively. Out of 192 colonies selected with this method, 38 were isolated and tested for their u.v.-sensitivity. Five of them showed significant, reproducible differences with respect to the parental line. As a partial characterization, the five u.v.-sensitive clones were assayed for unscheduled [3H]thymidine incorporation after exposure to u.v. light, by means of liquid scintillation spectrometry and autoradiography. In all clones. DNA repair synthesis was significantly decreased with respect to the parental line

  16. Brain-derived neurotrophic factor (BDNF) is required for the enhancement of hippocampal neurogenesis following environmental enrichment.

    Science.gov (United States)

    Rossi, Chiara; Angelucci, Andrea; Costantin, Laura; Braschi, Chiara; Mazzantini, Mario; Babbini, Francesco; Fabbri, Maria Elena; Tessarollo, Lino; Maffei, Lamberto; Berardi, Nicoletta; Caleo, Matteo

    2006-10-01

    Neurogenesis continues to occur in the adult mammalian hippocampus and is regulated by both genetic and environmental factors. It is known that exposure to an enriched environment enhances the number of newly generated neurons in the dentate gyrus. However, the mechanisms by which enriched housing produces these effects are poorly understood. To test a role for neurotrophins, we used heterozygous knockout mice for brain-derived neurotrophic factor (BDNF+/-) and mice lacking neurotrophin-4 (NT-4-/-) together with their wild-type littermates. Mice were either reared in standard laboratory conditions or placed in an enriched environment for 8 weeks. Animals received injections of the mitotic marker bromodeoxyuridine (BrdU) to label newborn cells. Enriched wild-type and enriched NT-4-/- mice showed a two-fold increase in hippocampal neurogenesis as assessed by stereological counting of BrdU-positive cells in the dentate gyrus and double labelling for BrdU and the neuronal marker NeuN. Remarkably, this enhancement of hippocampal neurogenesis was not seen in enriched BDNF+/- mice. Failure to up-regulate BDNF accompanied the lack of a neurogenic response in enriched BDNF heterozygous mice. We conclude that BDNF but not NT-4 is required for the environmental induction of neurogenesis. PMID:17040481

  17. A quantitative inverse relationship between connexin32 expression and cell proliferation in a rat hepatoma cell line

    International Nuclear Information System (INIS)

    Gap junctions comprised of connexin proteins are involved in direct intercellular communication and the regulation of cell behaviour and homeostasis. Reduced connexin expression and loss of gap junction function is a characteristic of many cancer cells and of the effect of many non-genotoxic carcinogens that induce cell proliferation. Moreover, when certain cancer cell lines are transfected with specific connexin genes, cells can regain control over proliferation. We have employed RNA interference and dexamethasone to modulate connexin32 expression in MH1C1 cells to a range of concentrations. This allowed the determination of the quantitative relationship between connexin32 protein expression and cell proliferation. The magnitude of cell proliferation, measured by bromodeoxyuridine incorporation, was inversely proportional to the level of connexin32 expression. Q-PCR indicated a lack of change of expression of a range of cell cycle-related genes at 24 h. The inverse relationship between Cx32 expression and proliferation was continuous, and a threshold level of reduction of connexin32 was not observable for an influence on proliferation

  18. Origin of Androgen-Insensitive Poorly Differentiated Tumors in the Transgenic Adenocarcinoma of Mouse Prostate Model

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    Wendy J. Huss

    2007-11-01

    Full Text Available Following castration, the transgenic adenocarcinoma of mouse prostate (TRAMP model demonstrates rapid development of SV40-Tag-driven poorly differentiated tumors that express neuroendocrine cell markers. The cell population dynamics within the prostates of castrated TRAMP mice were characterized by analyzing the incorporation of 5-bromodeoxyuridine (BrdUrd and the expression of SV40-Tag, synaptophysin, and androgen receptor (AR. Fourteen days postcastration, the remaining epithelial cells and adenocarcinoma cells were nonproliferative and lacked detectable SV40-Tag or synaptophysin expression. In contrast, morphologically distinct intraglandular foci were identified which expressed SV40-Tag, synaptophysin, and Ki67, but that lacked AR expression. These proliferative SV40-Tag and synaptophysin-expressing intraglandular foci were associated with the rare BrdUrd-retaining cells. These foci expanded rapidly in the postcastration prostate environment, in contrast to the AR- and SV40-Tag-expressing adenocarcinoma cells that lost SV40-Tag expression and underwent apoptosis after castration. Intraglandular foci of synaptophysin-expressing cells were also observed in the prostates of intact TRAMP mice at a comparable frequency; however, they did not progress to rapidly expanding tumors until much later in the life of the mice. This suggests that the foci of neuroendocrine-like cells that express SV40-Tag and synaptophysin, but lack AR, arise independent of androgen-deprivation and represent the source of the poorly differentiated tumors that are the lethal phenotype in the TRAMP model.

  19. Effect of Low Power Laser Irradiation on the Ability of Cell Growth and Myogenic Differentiation of Myoblasts Cultured In Vitro

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    Cui-Ping Zhang

    2014-01-01

    Full Text Available As a therapeutic modality, low power laser irradiation (LPLI has been used clinically in the treatment of skeletal muscle injuries and other myopathic conditions, but the cellular and molecular mechanisms attributed to this therapy were still unclear. Myoblasts are a type of myogenic stem cells quiescence in mature skeletal muscle fibers and are considered as the source cells during the regenerating process. The purpose of this paper was to investigate the effects of LPLI on the proliferation and myogenic differentiation of the cultured myoblasts and to find out the major candidates responsible for LPLI-induced muscle regeneration in vivo. In this study, primary rat myoblasts were exposed to helium-neon (He-Ne laser. Cell proliferation, differentiation, and the cellular responses to LPLI were monitored by using morphological observation and molecular biological methods. It was found that LPLI at a certain fluence could increase the cell growth potential for myoblasts and further induce more cells entering into S phase of the mitotic cycle as indicated by high levels of bromodeoxyuridine (BrdU incorporation, while at the same time inhibiting their in vitro differentiation and decreasing the expression of myogenic regulatory genes to a certain extent. Taken together, these results provide experimental evidence for the clinical applications of LPLI in regenerating skeletal muscle.

  20. Artesunate induces G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis in A431 human epidermoid carcinoma cells.

    Science.gov (United States)

    Jiang, Zhongyong; Chai, Jin; Chuang, Henry Hon Fung; Li, Shifeng; Wang, Tianran; Cheng, Yi; Chen, Wensheng; Zhou, Deshan

    2012-07-01

    The anticancer effects of artesunate (ART) have been well documented. However, its potential against skin cancer has not been explored yet. Herein we reported that 60 μmol/l ART effectively inhibited A431 (human epidermoid carcinoma cells) growth but not that of HaCaT (normal human keratinocyte cells). Our results revealed that ART induced cell cycle arrest at G0/G1 phase through the downregulation of cyclin A1, cyclin B, cyclin D1, Cdk2, Cdk4, and Cdk6. This correlated with the upregulation of p21 and p27. The 5-bromodeoxyuridine incorporation assay also indicated that ART treatment reduced DNA synthesis in a time-dependent manner. Furthermore, ART induced mitochondrial apoptosis, as evidenced by annexin V/propidium iodide staining and western blot analysis. Interestingly, ART-induced apoptosis diminished under iron-deficient conditions but intensified under iron-overload conditions. Taken together, these findings demonstrated the potential of ART in treating skin cancer through the induction of G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis and supported further investigations in other test systems. PMID:22421370

  1. Contribution of constitutively proliferating precursor cell subtypes to dentate neurogenesis after cortical infarcts

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    Oberland Julia

    2010-11-01

    Full Text Available Abstract Background It is well known that focal ischemia increases neurogenesis in the adult dentate gyrus of the hippocampal formation but the cellular mechanisms underlying this proliferative response are only poorly understood. We here investigated whether precursor cells which constitutively proliferate before the ischemic infarct contribute to post-ischemic neurogenesis. To this purpose, transgenic mice expressing green fluorescent protein (GFP under the control of the nestin promoter received repetitive injections of the proliferation marker bromodeoxyuridine (BrdU prior to induction of cortical infarcts. We then immunocytochemically analyzed the fate of these BrdU-positive precursor cell subtypes from day 4 to day 28 after the lesion. Results Quantification of BrdU-expressing precursor cell populations revealed no alteration in number of radial glia-like type 1 cells but a sequential increase of later precursor cell subtypes in lesioned animals (type 2a cells at day 7, type 3 cells/immature neurons at day 14. These alterations result in an enhanced survival of mature neurons 4 weeks postinfarct. Conclusions Focal cortical infarcts recruit dentate precursor cells generated already before the infarct and significantly contribute to an enhanced neurogenesis. Our findings thereby increase our understanding of the complex cellular mechanisms of postlesional neurogenesis.

  2. Early postnatal respiratory viral infection alters hippocampal neurogenesis, cell fate, and neuron morphology in the neonatal piglet.

    Science.gov (United States)

    Conrad, Matthew S; Harasim, Samantha; Rhodes, Justin S; Van Alstine, William G; Johnson, Rodney W

    2015-02-01

    Respiratory viral infections are common during the neonatal period in humans, but little is known about how early-life infection impacts brain development. The current study used a neonatal piglet model as piglets have a gyrencephalic brain with growth and development similar to human infants. Piglets were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) to evaluate how chronic neuroinflammation affects hippocampal neurogenesis and neuron morphology. Piglets in the neurogenesis study received one bromodeoxyuridine injection on postnatal day (PD) 7 and then were inoculated with PRRSV. Piglets were sacrificed at PD 28 and the number of BrdU+ cells and cell fate were quantified in the dentate gyrus. PRRSV piglets showed a 24% reduction in the number of newly divided cells forming neurons. Approximately 15% of newly divided cells formed microglia, but this was not affected by sex or PRRSV. Additionally, there was a sexual dimorphism of new cell survival in the dentate gyrus where males had more cells than females, and PRRSV infection caused a decreased survival in males only. Golgi impregnation was used to characterize dentate granule cell morphology. Sholl analysis revealed that PRRSV caused a change in inner granule cell morphology where the first branch point was extended further from the cell body. Males had more complex dendritic arbors than females in the outer granule cell layer, but this was not affected by PRRSV. There were no changes to dendritic spine density or morphology distribution. These findings suggest that early-life viral infection can impact brain development. PMID:25176574

  3. Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.

    Science.gov (United States)

    Haas, Christiane; Hegner, Richard; Helbig, Karsten; Bartels, Kristin; Bley, Thomas; Weber, Jost

    2016-06-01

    Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc. PMID:26614913

  4. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cell Therapy Promotes Functional Recovery of Contused Rat Spinal Cord through Enhancement of Endogenous Cell Proliferation and Oligogenesis

    Directory of Open Access Journals (Sweden)

    Sang In Park

    2012-01-01

    Full Text Available Numerous studies have shown the benefits of mesenchymal stem cells (MSCs on the repair of spinal cord injury (SCI model and on behavioral improvement, but the underlying mechanisms remain unclear. In this study, to investigate possible mechanisms by which MSCs contribute to the alleviation of neurologic deficits, we examined the potential effect of human umbilical cord blood-derived MSCs (hUCB-MSCs on the endogenous cell proliferation and oligogenesis after SCI. SCI was injured by contusion using a weight-drop impactor and hUCB-MSCs were transplanted into the boundary zone of the injured site. Animals received a daily injection of bromodeoxyuridine (BrdU for 7 days after treatment to identity newly synthesized cells of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells was evident. Behavior analysis revealed that locomotor functions of hUCB-MSCs group were restored significantly and the cavity volume was smaller in the MSCs-transplanted rats compared to the control group. In MSCs-transplanted group, TUNEL-positive cells were decreased and BrdU-positive cells were significantly increased rats compared with control group. In addition, more of BrdU-positive cells expressed neural stem/progenitor cell nestin and oligo-lineage cell such as NG2, CNPase, MBP and glial fibrillary acidic protein typical of astrocytes in the MSC-transplanted rats. Thus, endogenous cell proliferation and oligogenesis contribute to MSC-promoted functional recovery following SCI.

  5. Root cell patterning: a primary target for aluminium toxicity in maize.

    Science.gov (United States)

    Doncheva, Snezhanka; Amenós, Montserrat; Poschenrieder, Charlotte; Barceló, Juan

    2005-04-01

    The short-term influence (5-180 min) of 50 microM Al on cell division was investigated in root tips of two Zea mays L. varieties differing in Al-resistance. The incorporation of bromodeoxyuridine into S-phase nuclei was visualized by immunofluorescence staining using confocal laser fluorescence microscopy. In Al-sensitive plants 5 min Al exposure was enough to inhibit cell division in the proximal meristem (250-800 microm from the tip). After 10 or 30 min with Al only, a few S-phase nuclei were found in the cortical initials. By contrast, cell division was stimulated in the distal elongation zone (2.5-3.1 mm). After 180 min the protrusion of an incipient lateral root was observed in this zone. These observations suggest a fast change in cell patterning rather than a general cariotoxic effect after exposure to Al for a short time. No such changes were found in Al-resistant maize. This is the first report showing such fast Al-induced alterations in the number and the position of dividing cells in root tips. The observation that similar changes were induced by a local supply of naphthylphthalamic acid to the distal transition zone suggests that inhibition of auxin transport plays a role in the Al-induced alteration of root cell patterning. PMID:15737983

  6. DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz[a]anthracene 5,6-oxide

    International Nuclear Information System (INIS)

    Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz[a]anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification of bromodeoxyuridine photolysis employing the dye Hoechst 33258 and 365 nm radiation, and endonuclease-sensitive sites assay. Radioautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of the epoxide (approx. 10 μM) to be 0.1-0.2 that after a saturating ultraviolet dose (20 J/m2 at 254), though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive, although, considering the experimental errors, these data and those of repair replication are consistent with additivity. The epoxide did not inhibit loss of sites sensitive to the ultraviolet endonuclease. However, after a combined treatment to xeroderma pigmentosum cells there was appreciably less unscheduled synthesis than for the sum of both treatments and the epoxide inhibited the loss of nuclease-sensitive sites. (Auth.)

  7. Epigallocatechin-3-gallate rescues LPS-impaired adult hippocampal neurogenesis through suppressing the TLR4-NF-κB signaling pathway in mice.

    Science.gov (United States)

    Seong, Kyung-Joo; Lee, Hyun-Gwan; Kook, Min Suk; Ko, Hyun-Mi; Jung, Ji-Yeon; Kim, Won-Jae

    2016-01-01

    Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-κB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-κB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation. PMID:26807022

  8. Abca7 deletion does not affect adult neurogenesis in the mouse.

    Science.gov (United States)

    Li, Hongyun; Karl, Tim; Garner, Brett

    2016-01-01

    ATP-binding cassette transporter A7 (ABCA7) is highly expressed in the brain. Recent genome-wide association studies (GWAS) have identified ABCA7 single nucleotide polymorphisms (SNPs) that increase Alzheimer's disease (AD) risk, however, the mechanisms by which ABCA7 may control AD risk remain to be fully elucidated. Based on previous research suggesting that certain ABC transporters may play a role in the regulation of neurogenesis, we conducted a study of cell proliferation and neurogenic potential using cellular bromodeoxyuridine (BrdU) incorporation and doublecortin (DCX) immunostaining in adult Abca7 deficient mice and wild-type-like (WT) littermates. In the present study counting of BrdU-positive and DCX-positive cells in an established adult neurogenesis site in the dentate gyrus (DG) indicated there were no significant differences when WT and Abca7 deficient mice were compared. We also measured the area occupied by immunohistochemical staining for BrdU and DCX in the DG and the subventricular zone (SVZ) of the same mice and this confirmed that ABCA7 does not play a significant role in the regulation of cell proliferation or neurogenesis in the adult mouse. PMID:26792809

  9. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    Energy Technology Data Exchange (ETDEWEB)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui, E-mail: fuyh@fudan.edu.cn

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  10. Effect of bone marrow derived mesenchymal stem cells on lung pathology and inflammation in ovalbumin-induced asthma in mouse

    Directory of Open Access Journals (Sweden)

    Maryam Mohammadian

    2016-01-01

    Full Text Available Objective(s:Bone marrow-derived mesenchymal stem cells (BMSCs have attracted significant interest to treat asthma and its complication. In this study, the effects of BMSCs on lung pathology and inflammation in an ovalbumin-induced asthma model in mouse were examined. Materials and Methods:BALB/c mice were divided into three groups: control group (animals were not sensitized, asthma group (animals were sensitized by ovalbumin, asthma+BMSC group (animals were sensitized by ovalbumin and treated with BMSCs. BMSCs were isolated and characterized and then labeled with Bromodeoxyuridine (BrdU. After that the cells transferred into asthmatic mice. Histopathological changes of the airways, BMSCs migration and total and differential white blood cell (WBC count in bronchoalveolar lavage (BAL fluid were evaluated. Results:A large number of BrdU-BMSCs were found in the lungs of mice treated with BMSCs. The histopathological changes, BAL total WBC counts and the percentage of neutrophils and eosinophils were increased in asthma group compared to the control group. Treatment with BMSCs significantly decreased airway pathological indices, inflammatory cell infiltration, and also goblet cell hyperplasia. Conclusion:The results of this study revealed that BMSCs therapy significantly suppressed the lung pathology and inflammation in the ovalbumin induced asthma model in mouse.

  11. Therapeutic Potential of Umbilical Cord Blood Stem Cells on Brain Damage of a Model of Stroke

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Nikravesh

    2011-11-01

    Full Text Available Introduction: Human cord blood-derived stem cells are a rich source of stem cells as well as precursors. With regard to the researchers have focused on the therapeutic potential of stem cell in the neurological disease such as stroke, the aim of this study was the investiga-tion of the therapeutic effects of human cord blood-derived stem cells in cerebral ischemia on rat. Methods: This study was carried out on young rats. Firstly, to create a laboratory model of ischemic stroke, carotid artery of animals was occluded for 30 minutes. Then, umbilical cord blood cells were isolated and labeled using bromodeoxyuridine and 2×105 cells were injected into the experimental group via the tail vein. Rats with hypoxic condi-tions were used as a sham group. A group of animals did not receive any injection or sur-geries were used as a control. Results: Obtained results were evaluated based on behavior-al responses and immunohistochemistry, with emphasis on areas of putamen and caudate nucleus in the control, sham and experimental groups. Our results indicated that behavioral recovery was observed in the experimental group compared to the either the sham or the control group. However, histological studies demonstrated a low percent of tissue injury in the experimental group in comparison with the sham group. Conclusion: Stem cell trans-plantation is beneficial for the brain tissue reparation after hypoxic ischemic cell death.

  12. Mitosis orientation in prostate epithelial cells changed by endocrine effect

    Institute of Scientific and Technical Information of China (English)

    Xiang-yun LIU; Dong-mei Li; Xiao-fang ZHANG; Jian-hui WU; Zu-yue SUN

    2008-01-01

    Aim: The aim of the present study was to investigate the effect of androgen and estrogen on mitosis orientation in the prostate epithelial cells of male rats. Methods: Castrated rats were treated with a single injection of testosterone propionate (TP) or benzogynestry (E2). There were 8 rats in the control group and TP-treated or E2-treated group. Prostate, liver, a specimen of skin, and a segment of the jejunum and colon were removed after the corresponding treatment. The results were observed through immunohistochemistry and iron hematoxylin-eosin staining.Results: All mitoses found in the prostate epithelial cells of castrated rats with TP were oriented parallel to the basement membrane; however, mitoses found in the prostate epithelial cells of castrated rats in E2 and the control group were oriented perpendicular to the basement membrane. TP treatment resulted in marked changes in mitosis orientation in the prostate epithelial cells. Bromodeoxyuridine-labeled positive cells could be seen throughout the stroma and prostate epithelial cells with an injection of TP; however, the positive cells could only be seen in the stroma of prostate with an injection of E2, and the positive cells could hardly be seen in the control group. Conclusion: We found a novel effect of TP in the prostate as a marked change of mitosis orientation in prostate epithelial cells.

  13. Evaluation of silicon nitride as a substrate for culture of PC12 cells: an interfacial model for functional studies in neurons.

    Directory of Open Access Journals (Sweden)

    Johan Jaime Medina Benavente

    Full Text Available Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.

  14. Immune response in mice and cattle after immunization with a Boophilus microplus DNA vaccine containing bm86 gene.

    Science.gov (United States)

    Ruiz, Lina María; Orduz, Sergio; López, Elkin D; Guzmán, Fanny; Patarroyo, Manuel E; Armengol, Gemma

    2007-03-15

    Plasmid pBMC2 encoding antigen Bm86 from a Colombian strain of cattle tick Boophilus microplus, was used for DNA-mediated immunization of BALB/c mice, employing doses of 10 and 50microg, delivered by intradermic and intramuscular routes. Anti-Bm86 antibody levels were significantly higher compared to control mice treated with PBS. In the evaluation of immunoglobulin isotypes, significant levels of IgG2a and IgG2b were observed in mice immunized with 50microg of pBMC2. Measurement of interleukine (IL) levels (IL-4, IL-5, IL-12(p40)) and interferon-gamma (IFN-gamma) in the sera of mice immunized with pBMC2 indicated high levels of IL-4 and IL-5, although there were also significant levels of IFN-gamma. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN-gamma secretion. In all trials, mice injected intramuscularly with 50microg of pBMC2 presented the highest immune response. Moreover, cattle immunized with this DNA vaccine showed antibody production significantly different to the negative control. In conclusion, these results suggest the potential of DNA immunization with pBMC2 to induce humoral and cellular immune responses against B. microplus. PMID:17055651

  15. Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1

    Directory of Open Access Journals (Sweden)

    Hyang Jee

    2013-01-01

    Full Text Available Gap junctions and their structural proteins, connexins (Cxs, havebeen implicated in carcinogenesis. To explore the involvement ofCx32 in gastric carcinogenesis, immunochemical analysis of Cx32and proliferation marker Ki67 using tissue-microarrayed humangastric cancer and normal tissues was performed. In addition, afterCx32 overexpression in the human gastric cancer cell line AGS,cell proliferation, cell cycle analyses, and p21Cip1 and p27Kip1expression levels were examined by bromodeoxyuridine assay,flow cytometry, real-time RT-PCR, and western blotting.Immunohistochemical study noted a strong inverse correlationbetween Cx32 and Ki67 expression pattern as well as theirlocation. In vitro, overexpression of Cx32 in AGS cells inhibitedcell proliferation significantly. G1 arrest, up-regulation of cellcycle-regulatory proteins p21Cip1 and p27Kip1 was also found atboth mRNA and protein levels. Taken together, Cx32 plays someroles in gastric cancer development by inhibiting gastric cancercell proliferation through cell cycle arrest and cell cycle regulatoryproteins. [BMB Reports 2013; 46(1: 25-30

  16. Maternal immune activation differentially impacts mature and adult-born hippocampal neurons in male mice.

    Science.gov (United States)

    Zhang, Zhi; van Praag, Henriette

    2015-03-01

    Schizophrenia is associated with deficits in the hippocampus, a brain area important for learning and memory. The dentate gyrus (DG) of the hippocampus develops both before and after birth. To study the relative contribution of mature and adult-born DG granule cells to disease etiology, we compared both cell populations in a mouse model of psychiatric illness resulting from maternal immune activation. Polyriboinosinic-polyribocytidilic acid (PolyIC, 5mg/kg) or saline was given on gestation day 15 to pregnant female C57Bl/6 mice. Male offspring (n=105), was administered systemic bromodeoxyuridine (BrdU, 50mg/kg) (n=52) or intracerebral retroviral injection into the DG (n=53), to label dividing cells at one month of age. Two months later behavioral tests were performed to evaluate disease phenotype. Immunohistochemistry and whole-cell patch clamping were used to assess morphological and physiological characteristics of DG cells. Three-month-old PolyIC exposed male offspring exhibited deficient pre-pulse inhibition, spatial maze performance and motor coordination, as well as increased depression-like behavior. Histological analysis showed reduced DG volume and parvalbumin positive interneuron number. Both mature and new hippocampal neurons showed modifications in intrinsic properties such as increased input resistance and lower current threshold, and decreased action potential number. Reduced GABAergic inhibitory transmission was observed only in mature DG neurons. Differential impairments in mature DG cells and adult-born new neurons may have implications for behavioral deficits associated with maternal immune activation. PMID:25449671

  17. BrdU Pulse Labelling In Vivo to Characterise Cell Proliferation during Regeneration and Repair following Injury to the Airway Wall in Sheep

    Directory of Open Access Journals (Sweden)

    B. Yahaya

    2013-01-01

    Full Text Available The response of S-phase cells labelled with bromodeoxyuridine (BrdU in sheep airways undergoing repair in response to endobronchial brush biopsy was investigated in this study. Separate sites within the airway tree of anaesthetised sheep were biopsied at intervals prior to pulse labelling with BrdU, which was administered one hour prior to euthanasia. Both brushed and spatially disparate unbrushed (control sites were carefully mapped, dissected, and processed to facilitate histological analysis of BrdU labelling. Our study indicated that the number and location of BrdU-labelled cells varied according to the age of the repairing injury. There was little evidence of cell proliferation in either control airway tissues or airway tissues examined six hours after injury. However, by days 1 and 3, BrdU-labelled cells were increased in number in the airway wall, both at the damaged site and in the regions flanking either side of the injury. Thereafter, cell proliferative activity largely declined by day 7 after injury, when consistent evidence of remodelling in the airway wall could be appreciated. This study successfully demonstrated the effectiveness of in vivo pulse labelling in tracking cell proliferation during repair which has a potential value in exploring the therapeutic utility of stem cell approaches in relevant lung disease models.

  18. Chlorella vulgaris triggers apoptosis in hepatocarcinogenesis-induced rats

    Institute of Scientific and Technical Information of China (English)

    Emey Suhana MOHD AZAMAI; Suhaniza SULAIMAN; Shafina Hanim MOHD HABIB; Mee Lee LOOI; Srijit DAS; Nor Aini ABDUL HAMID; Wan Zurinah WANG NGAH; Yasmin Anum MOHD YUSOF

    2009-01-01

    Chlorella vulgaris (CV) has been reported to have antioxidant and anticancer properties. We evaluated the effect of CV on apoptotic regulator protein expression in liver cancer-induced rats. Male Wistar rats (200-250 g) were divided into eight groups: control group (normal diet), CDE group (choline deficient diet supplemented with ethionine in drinking water to induce hepatocarcinogenesis), CV groups with three different doses of CV (50, 150, and 300 mg/kg body weight), and CDE groups treated with different doses of CV (50, 150, and 300 mg/kg body weight). Rats were sacrificed at various weeks and liver tissues were embedded in paraffin blocks for immunohistochemistry studies. CV, at increasing doses, decreased the expression of anti-apoptotic protein, Bcl-2, but increased the expression of pro-apoptotic protein, caspase 8, in CDE rats, which was correlated with decreased hepatoctyes proliferation and increased apoptosis as determined by bromodeoxy-uridine (BrdU) labeling and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay, respectively. Our study shows that CV has definite chemopreventive effect by inducing apoptosis via decreasing the expression of Bcl-2 and increasing the expression of caspase 8 in hepatocarcinogenesis-induced rats.

  19. Cadmium is more toxic to LLC-PK1 cells than to MDCK cells acting on the cadherin-catenin complex.

    Science.gov (United States)

    Zimmerhackl, L B; Momm, F; Wiegele, G; Brandis, M

    1998-07-01

    Cadmium toxicity to renal cells was investigated in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells as models of the distal tubule/collecting duct and proximal tubule, respectively. Cells were grown on two-compartment filters and exposed to 0.1-50 microM Cd2+. In MDCK cells, Cd2+ was more toxic from the basolateral than from the apical side and dependent on the extracellular Ca2+ concentration. Toxicity was evident within 24 h, as shown by a decrease in transepithelial resistance (TER), reduced proliferation (bromodeoxyuridine incorporation), reduction in ATP concentration, and morphological changes. On confocal microscopy, E-cadherin and alpha-catenin staining patterns indicated interference with the cadherin-catenin complex. LLC-PK1 cells showed a similar toxicity pattern, which was evident at lower Cd2+ concentrations. An increase of E-cadherin and alpha-catenin molecules in the Triton X-100-insoluble fraction was detectable at high Cd2+ concentrations in LLC-PK1 cells but not in MDCK cells. Lactate dehydrogenase release indicated membrane leakage in LLC-PK1 cells. Rhodamine-phalloidin staining, a probe for F-actin filaments, demonstrated alterations of the actin cytoskeleton in both cell lines. In conclusion, cadmium caused ATP depletion and interfered with the cadherin-catenin complex and probably the tight junctions changing renal cell morphology and function. PMID:9689016

  20. Effects of exercise on neurogenesis in the dentate gyrus and ability of learning and memory after hippocampus lesion in adult rats

    Institute of Scientific and Technical Information of China (English)

    Lin CHEN; Shan GONG; Li-Dong SHAN; Wei-Ping XU; Yue-Jin ZHANG; Shi-Yu GUO; Tadashi Hisamitsu; Qi-Zhang YIN; Xing-Hong JIANG

    2006-01-01

    Objective To explore the effects of exercise on dentate gyrus (DG) neurogenesis and the ability of learning and memory in hippocampus-lesioned adult rats. Methods Hippocampus lesion was produced by intrahippocampal microinjection of kainic acid (KA). Bromodeoxyuridine (BrdU) was used to label dividing cells. Y maze test was used to evaluate the ability of learning and memory. Exercise was conducted in the form of forced running in a motor-driven running wheel. The speed of wheel revolution was regulated at 3 kinds of intensity: lightly running, moderately running, or heavily running. Results Hippocampus lesion could increase the number of BrdU-labeled DG cells, moderately running after lesion could further enhance the number of BrdU-labeled cells and decrease the error number (EN) in Y maze test,while neither lightly running, nor heavily running had such effects. There was a negative correlation between the number of DG BrdU-labeled cells and the EN in the Y maze test after running. Conclusion Moderate exercise could enhance the DG neurogenesis and ameliorate the ability of learning and memory in hippocampus-lesioned rats.

  1. Nephroprotective effect of bee honey and royal jelly against subchronic cisplatin toxicity in rats.

    Science.gov (United States)

    Ibrahim, Abdelazim; Eldaim, Mabrouk A Abd; Abdel-Daim, Mohamed M

    2016-08-01

    Cisplatin is one of the most potent and effective chemotherapeutic agents. However, its antineoplastic use is limited due to its cumulative nephrotoxic side effects. Therefore, the present study was undertaken to examine the nephroprotective potential of dietary bee honey and royal jelly against subchronic cisplatin toxicity in rats. Male Wistar rats were randomly divided into controls, cisplatin-treated, bee honey-pretreated cisplatin-treated and royal jelly-pretreated cisplatin-treated groups. Bee honey and royal jelly were given orally at doses of 20 and 100 mg/kg, respectively. Subchronic toxicity was induced by cisplatin (1 mg/kg bw, ip), twice weekly for 10 weeks. Cisplatin treated animals revealed a significant increase in serum level of renal injury products (urea, creatinine and uric acid). Histopathologically, cisplatin produced pronounced tubulointerstitial injuries, upregulated the fibrogenic factors, α-smooth muscle actin (α-SMA) and transforming growth factor β1(TGF-β1), and downregulated the cell proliferation marker, bromodeoxyuridine (Brdu). Dietary bee honey and royal jelly normalized the elevated serum renal injury product biomarkers, improved the histopathologic changes, reduced the expression of α-SMA and TGF-β1 and increased the expression of Brdu. Therefore, it could be concluded that bee honey, and royal jelly could be used as dietary preventive natural products against subchronic cisplatin-induced renal injury. PMID:25720368

  2. In vivo cell kinetic measurements in a randomized trial of continuous hyperfractionated accelerated radiotherapy with or without mitomycin C in head-and-neck cancer

    International Nuclear Information System (INIS)

    Purpose: Tumor cell repopulation is still considered to be a major cause of failure in radiotherapy. In this study, we investigated the influence of cell kinetic parameters on the outcome of patients treated in a randomized trial of accelerated fractionation, with or without mitomycin C, vs. conventional fractionation. Methods and Materials: Sixty-two patients were studied using administration of bromodeoxyuridine (BrdUrd), and cell kinetic parameters were measured using flow cytometry. The patients were treated with either 70 Gy for 7 weeks or 55.3 Gy for 17 continuous days (V-CHART) with or without 20 mg/m2 mitomycin C on day 5. Results: The potential doubling time (Tpot) and labeling index (LI) failed to provide any prognostic information with regard to local control or survival. However, the duration of the S phase (Ts) revealed patients whose tumors had a long Ts had significantly worse local control (p = 0.028) and survival (p = 0.034) irrespective of treatment. A similar trend was evident within the different treatment arms particularly associated with overall survival. Conclusions: The Ts values of head-and-neck squamous cell cancers provided prognostic information that predicted clinical outcome irrespective of treatment schedule in this study. This neglected parameter of the Tpot method might provide information related to redistribution of cells during fractionated radiotherapy

  3. Somatic genomic variations in extra-embryonic tissues

    Energy Technology Data Exchange (ETDEWEB)

    Weier, Jingly F.; Ferlatte, Christy; Weier, Heinz-Ulli G.

    2010-05-21

    In the mature chorion, one of the membranes that exist during pregnancy between the developing fetus and mother, human placental cells form highly specialized tissues composed of mesenchyme and floating or anchoring villi. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes; however, chromosome losses were detected infrequently. With chromosomes gained in what appeared to be a chromosome-specific manner, more than half of the invasive cytotrophoblasts in normal pregnancies were found to be hyperdiploid. Interestingly, the rates of hyperdiploid cells depended not only on gestational age, but were strongly associated with the extraembryonic compartment at the fetal-maternal interface from which they were isolated. Since hyperdiploid cells showed drastically reduced DNA replication as measured by bromodeoxyuridine incorporation, we conclude that aneuploidy is a part of the normal process of placentation potentially limiting the proliferative capabilities of invasive cytotrophoblasts. Thus, under the special circumstances of human reproduction, somatic genomic variations may exert a beneficial, anti-neoplastic effect on the organism.

  4. Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients.

    Science.gov (United States)

    Miller, M A; Mazewski, C M; Yousuf, N; Sheikh, Y; White, L M; Yanik, G A; Hyams, D M; Lampkin, B C; Raza, A

    1991-04-01

    Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors. PMID:2005370

  5. Cell proliferation during fractionated radiation in two experimental tumors

    International Nuclear Information System (INIS)

    Tumor cell proliferation kinetics after irradiation have been studied using the method of bromodeoxyuridine incorporation and flow cytometry. Labelling indices were obtained after single and multiple fractions of radiation in a mouse fibrosarcoma (FSa-II) and human squamous carcinoma (FADU) growing in nude mice. For 8 mm tumors mean L.I was 17.5 +- 2.6% and 21.5 + 3.2%, respectively. Both tumors showed a similar response to single dose of irradiation (10 and 20 Gy) with initial depression of labelling index and then a rapid increase after 3 days in the FSa-II tumors (mean L.I 24%) and 5 days in the FADU tumors (mean L.I 27%). During fractionated treatment, labelling index was dependent on dose per fraction (2.5-18 Gy) time interval between fractions and time of analysis. Tumors were biopsied during course of fractionated treatment to see if labelling index would act as a predictor of response. No significant difference could be determined between individual tumors that had received the same dose per fraction. However a labelling index the same or higher than control values were associated with lack of tumor control. Controlled tumors showed a significant depression of labelling index (rho<0.05)

  6. Olmesartan inhibits the expression of monocyte chemoattractant protein-1 and tumor necrosis factor-α and improves vascular remodeling after vascular injury in mouse

    Institute of Scientific and Technical Information of China (English)

    李震; 陈小东; 倪少凯; 李建文; 林木生

    2004-01-01

    Objective: To investigate the neointima formation and the expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in cuff-induced vascular injury in mouse model, and to examine the effect of angiotensin II type 1 receptor (AT1) blocker, olmesartan, on MCP-1 and TNF-α expression and consequently vascular remodeling.Methods: Vascular injury was induced by polyethylene cuff-placement around the mouse femoral artery. Some mice were treated with AT1 receptor blocker, olmesartan, at the dose of 3 mg*kg-1*day-1 with an osmotic minipump. Neointima formation and the proliferation of vascular smooth muscle cells (VSMCs) were measured by morphometric analysis and bromodeoxyuridine (BrdU) incorporation. MCP-1 and TNF-α expression was detected by Western blot and immunohistochemical staining.Results: We observed neointima formation 14 days after cuff placement as well as VSMCs proliferation in the media and neointima. Cuff placement also induced MCP-1 and TNF-α expression in the media and neointima that the VSMCs specifically existed. Treatment of mice with olmesartan at a dose of 3 mg*kg-1*day-1, which did not influence systolic blood pressure, significantly decreased neointima formation and the proliferation of VSMCs. Olmesartan also inhibited MCP-1 and TNF-α expression in the injured arteries.Conclusions: Our results demonstrate that blockade of AT1 receptor inhibits MCP-1 and TNF-α expression and thereby improves vascular remodeling.

  7. 信息动态%Evaluation of Mitochondrial Damage of lsletβCells by Mitochondrial Permeability Transition Pore

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To evaluate the mitochondrial damage of islet β cells under glucolipotoxicity by investigating the mitochondrial permeability transition pore (mPTP). Methods Pancreatic β cell lines INS-1 cells were treated with 0. 4 mmol/L palmitic acid and different concentrations of glucose (5.6 mmol/L or 25 mmol/L). The mitochondrial membrane potential, mPTP and reactive oxygen species (ROS) were measured by flow cytometry and fluorescence staining technique to assess the mitochon drial damage. Cell proliferation was measured by 5-bromodeoxyuridine incorporation and cell apoptosis was detected by Annexin V method. Results Compared with the low glucose concentration, the high glucose concentration resulted in decreased mPTP activity (P<0.05), increased mitochondrial membrane potential (P<0.05) and increased cell proliferation rate (P<0.05). There was no significant change in ROS generation. When cells were exposed to high glucose concentration and palmitic acid, both mPTP activity and mitochonhdrial membrane potential reduced (P<0.05), with increased cell apoptosis rate (P <0.05) and increased ROS generation. Conclusion The high glucose concentration decreases mPTP and increases mitochondrial membrane potential, suggesting that cells may remain in an unstable high metabolic state. Evaluation of mPTP may contribute to a more comprehensive understanding of mitochondrial dysfunction under glucotoxictiy.

  8. Relative biological effectiveness of carbon ions for causing fatal liver failure after partial hepatectomy in mice

    International Nuclear Information System (INIS)

    To evaluate the acute phase damage to liver by carbon ions, BALB/c mice were irradiated with carbon ions or X-rays after two-thirds partial hepatectomy, and their survival was followed. The 50% lethal dose within 60 days (LD50/60) was 42.2±0.25 Gy (standard error) for X-rays, and 22.7±0.25 Gy for carbon ions. The relative biological effectiveness (RBE) of carbon ions was 1.86 (95% confident limits: 1.69-2.04) as calculated from the LD50/60. Mice irradiated at much higher doses, 60 Gy of X-rays or 24 Gy of carbon ions, showed significantly higher serum ammonia levels and lower serum albumin levels than normal, suggesting hepatic failure as a cause of death. Hepatocytes showed karyorrhexis and karyolysis in carbon ion irradiated and spotty necrosis in X-ray irradiated mice, suggesting nuclear damage. Mice irradiated with LD50 of X-rays or carbon ions had a remarkably lower bromodeoxyuridine (BrdU) labeling index and mitotic index than control. Treatments with both BrdU and vincristine showed that none of the hepatocytes that synthesized DNA after irradiation completed mitosis, indicating G2 arrest. The liver weight of irradiated mice significantly decreased depending on the dose. Carbon ions as well as X-rays damaged hepatocytes directly and suppressed liver regeneration leading to fatal liver failure. (author)

  9. Radiation-induced depression of DNA synthesis in cultured mammalian cells

    International Nuclear Information System (INIS)

    A 313-nm light source was constructed in order to study the mechanisms by which ultraviolet and ionizing radiations inhibit DNA synthesis. It was found that in CHO, MDBK and HeLa cells, grown for one generation in the DNA sensitizer bromodeoxyuridine (BrdUrd), 313-nm light inhibited DNA synthesis with a pattern similar to that of the effect of x-rays on normal cells. A biphasic dose response curve for inhibition of total synthesis was observed, with a sensitive component representing depression of initiation of new replicons and a resistant component representing interference with elongation of replicons already growing at the time of irradiation. Since the BrdUrd plus 313-nm light treatment produces DNA lesions similar to those produced by x-rays (base damage, strand breaks, crosslinks) these results suggest that the effect of x-rays on DNA synthesis is mediated by DNA damage. In experiments with synchronized cells, it was found that in cells in which about half the chromosomes had incorporated BrdUrd, 313-nm light inhibited replication of the BrdUrd-containing DNA, but had no effect on the replication of the unsubstituted DNA in the same cell. Thus the information that DNA is damaged appears to be propagated along the DNA molecule from the sites of damage to the replication initiation sites as some kind of conformational change, possibly a relaxation of superhelical tension. Target theory calculations suggest that a single DNA lesion prevents the initiation of several adjacent replicons

  10. Fatherhood reduces the survival of adult-generated cells and affects various types of behavior in the prairie vole (Microtus ochrogaster ).

    Science.gov (United States)

    Lieberwirth, Claudia; Wang, Yue; Jia, Xixi; Liu, Yan; Wang, Zuoxin

    2013-11-01

    Motherhood has profound effects on physiology, neuronal plasticity, and behavior. We conducted a series of experiments to test the hypothesis that fatherhood, similarly to motherhood, affects brain plasticity (such as cell proliferation and survival) and various behaviors in the highly social prairie vole (Microtus ochrogaster). In Experiment 1, adult males were housed with their same-sex cage mate (control), single-housed (isolation), or housed with a receptive female to mate and produce offspring (father) for 6 weeks. Fatherhood significantly reduced cell survival (assessed by bromodeoxyuridine labeling), but not cell proliferation (assessed by Ki67-labeling), in the amygdala, dentate gyrus of the hippocampus, and ventromedial hypothalamus, suggesting that fatherhood affects brain plasticity. In Experiment 2, neither acute (20 min) nor chronic (20 min daily for 10 consecutive days) pup exposure altered cell proliferation or survival in the brain, but chronic pup exposure increased circulating corticosterone levels. These data suggest that reduced cell survival in the brain of prairie vole fathers was unlikely to be due to the level of pup exposure and display of paternal behavior, and may not be mediated by circulating corticosterone. The effects of fatherhood on various behaviors (including anxiety-like, depression-like, and social behaviors) were examined in Experiment 3. The data indicated that fatherhood increased anxiety- and depression-like behaviors as well as altered aggression and social recognition memory in male prairie voles. These results warrant further investigation of a possible link between brain plasticity and behavioral changes observed due to fatherhood. PMID:23899240

  11. Bcl-2 enhances the formation of newborn striatal long-projection neurons in adult rat brain after a transient ischemic stroke

    Institute of Scientific and Technical Information of China (English)

    Jian-Jun Guo; Fang Liu; Xiao Sun; Jun-Jie Huang; Ming Xu; Feng-Yan Sun

    2012-01-01

    Objective It has been reported that B-cell lymphoma 2 (Bcl-2) enhances neurogenesis as well as supporting axonal growth after injury.In the present study,we investigated whether Bcl-2 overexpression plays a role in the formation of newborn striatonigral projection neurons in the adult rat brain after transient middle cerebral artery occlusion (MCAO).Methods We infused human Bcl-2-expressing plasmid (pBcl-2) into the lateral ventricle immediately after 30 min of MCAO,injected 5'-bromodeoxyuridine (BrdU) intraperitoneally to label proliferative cells,and microinjected fluorogold (FG) into the substantia nigra at 11 weeks of reperfusion followed by multiple immunostaining of striatonigral projection neurons at 12 weeks.Results We found that pBcl-2 treatment significantly increased the number of newborn neurons (BrdU+-NeuN+) in the striatum ipsilateral to the MCAO.We further detected newborn striatonigral projection neurons (BrdU+-FG+-NeuN+) in the ipsilateral striatum at 12 weeks.More interestingly,the number of newborn striatonigral projection neurons (BrdU+-FG+) was significantly increased by pBcl-2 treatment compared to that by pEGFP,a control plasmid.Conclusion Taken together,we found that Bcl-2 overexpression in the brain enhanced the generation of newborn striatonigral projection neurons.This provides a potential strategy for promoting the reestablishment of neural networks and brain repair after ischemic injury.

  12. The localization and uptake of in ovo injected soluble and particulate substances in the chicken.

    Science.gov (United States)

    Jochemsen, P; Jeurissen, S H M

    2002-12-01

    The localization of in ovo injected substances in the chicken was investigated. We determined that localization is dependent on the nature of substances and the time of in ovo injection. In ovo injections with soluble bromodeoxyuridin (BrdU), particulate colloidal carbon, 40 nm fluorescent microspheres, and live Infectious Bursal Disease Virus (IBDV) were performed with a 25-mm (1-in) needle at Days 16 and 18 of incubation (DI-16 and DI-18, respectively). Localization of injected substances was determined in several organs using immunocytochemical methods. At DI-16, approximately 50% of the substances were detected in the organs; therefore, the localization of substances was not consistent. At DI-18, the substances were injected into the amnion. The substances entered the embryo by the mouth and were ingested into the intestinal and respiratory tract. All substances reached the lungs of the embryo via the trachea and the bronchi and were absorbed by the gas exchange tissue. In addition, the substances were absorbed by the bursa. Particulate colloidal carbon and microspheres remained in the organs where they were taken up initially for the rest of time of the experiment. Live IBDV, however, was distributed to other organs of the embryo. Soluble BrdU was found in all investigated organs of the embryo in high amounts. These results demonstrate that in ovo injection at DI-18 is an effective route to introduce substances into the chicken embryo, whereby the characteristics of the substance determine its final localization. PMID:12512571

  13. Effects of hyperthermia and x irradiation on sister chromatid exchange (SCE) frequency in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    The BrdUrd labeling method was used to evaluate the effects of hyperthermia, x irradiation, and the combined treatment on the incidence of sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells. Cells cultured in McCoy's 5A media containing 10 μM 5-bromodeoxyuridine were synchronized after one cell cycle by mitotic shake-off. Early-G1 cells were heated by submerging culture flasks in a 44 +- 0.050C water bath for periods of 20, 40, and 60 min. By the same method, other cultures were x irradiated at doses of 100, 200, 400, and 600 rad. A third protocol involved combined treatment of 20 min at 440C followed immediately by one of the above radiation doses. A fourth protocol reversed the sequence of the combined treatment applying x irradiation (200 or 400 rad) followed immediately by hyperthermia. The data showed that hyperthermia and x irradiation both elevated the frequency of SCEs significantly whether applied separately or together. The combined treatment (heat: 20 min at 440C plus varying x-radiation doses) produced results suggestive of a synergistic interaction. The sequence of the heat and x irradiation did not appear to have a significant effect on the production of SCE

  14. Requirement of mitoses for the reversal of X-inactivation in cell hybrids between murine embryonal carcinoma cells and normal female thymocytes

    International Nuclear Information System (INIS)

    By means of a 5-bromodeoxyuridine (BrdU) incorporation and acridine orange fluorescence staining method the authors studied reactivation of the inactivated X chromosome (Xi) in newly formed cell hybrids between the near-diploid HPRT-deficient OTF9-63 murine embryonal carcinoma cell (ECC) with an XO sex chromosome constitution and the normal female mouse thymocyte. Synchronization of the late replicating S chromosome in such hybrid cells, indicative of reactivation, was found for the first time on Day 3, and the frequency of reactivation was attained 90% on Day 5. Inhibition of cell cycle progression either by methylglyoxal bis(guanylhydrazone) dihydrochloride, an inhibitor of polyamine metabolism, or by isoleucine-deficient medium after cell fusion delayed reactivation of the Xi, which implied that the number of cell division cycles traversed by individual cells rather than the length of time after cell fusion is critical for the reactivation. Double-labeling experiments using [3H]thymidine and BrdU indicated that hybrid cells had undergone three or four mitoses before reactivation of the Xi. Most probably reactivation of the Xi is consequent to reversion of the thymocyte genome to an undifferentiated state under the influence of OTF9 genome. DNA demethylation or dilution of Xi-specific factors by mitoses may be involved in this process

  15. Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A.

    Science.gov (United States)

    Miró, Lluïsa; Pérez-Bosque, Anna; Maijó, Mònica; Naftalin, Richard J; Moretó, Miquel

    2014-10-01

    Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factor A (PDGFA), platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism. PMID:25085844

  16. Constraint-induced movement therapy enhanced neurogenesis and behavioral recovery after stroke in adult rats.

    Science.gov (United States)

    Zhao, Chuansheng; Wang, Jun; Zhao, Shanshan; Nie, Yingxue

    2009-08-01

    Constraint-induced movement therapy (CIMT) has been extensively used for stroke rehabilitation. CIMT encourages use of the impaired limb along with restraint of the ipsilesional limb in daily life, and may promote behavioral recovery and induce structural changes in brain after stroke. The aim of this study was to investigate whether CIMT enhances neurogenesis in rat brain after stroke that was generated by middle cerebral artery occlusion. Adult rats were divided into sham group, ischemia group and ischemia treated with CIMT group. Rats of CIMT group were treated with a plaster cast to restrain the healthy forelimb for 14 days beginning 1 week after ischemia. The proliferation of neuronal cells labeled with bromodeoxyuridine (BrdU) and behavioral recovery were analyzed at day 29 after ischemia. We also measured the tissue level of stromal cell-derived factor 1 (SDF-1) by ELISA. SDF-1 might be involved in the regulation of neurogenesis following stroke. In the subventricular zone of the animals treated with CIMT, there was a significant increase in the number of BrdU-positive cells (135 +/- 18, P behavioral performances and increased the SDF-1 protein levels in the cortex and dentate gyrus. In conclusion, CIMT treatment enhances neurogenesis and functional recovery after stroke. PMID:19638734

  17. Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury

    DEFF Research Database (Denmark)

    Ladeby, Rune; Wirenfeldt, Martin; Dalmau, Ishar;

    2005-01-01

    following transection of the entorhino-dentate perforant path projection. To investigate the possible link between microglia and hematopoietic precursors, we analyzed the expression of the stem cell marker CD34 by lesion-reactive microglia in conjunction with the proliferation marker bromodeoxyuridine (Brd......U) and the use of radiation bone marrow (BM) chimeric mice. We found that CD34 is upregulated on early-activated resident microglia, rather than by infiltrating bone marrow-derived cells. The number of CD34(+) microglia peaked at day 3 when 67% of the resident CD11b/Mac-1(+) microglia co-expressed CD34......, and all CD34(+) cells co-expressed Mac-1, and decreased sharply toward day 5, unlike Mac-1, which was maximally expressed at day 5. Approximately 80% of the CD34(+) cells in the denervated dentate gyrus had incorporated BrdU into their nuclei at day 3. We also showed that CD34 is upregulated on early...

  18. Carbon Nanohorns Promote Maturation of Neonatal Rat Ventricular Myocytes and Inhibit Proliferation of Cardiac Fibroblasts: a Promising Scaffold for Cardiac Tissue Engineering.

    Science.gov (United States)

    Wu, Yujing; Shi, Xiaoli; Li, Yi; Tian, Lei; Bai, Rui; Wei, Yujie; Han, Dong; Liu, Huiliang; Xu, Jianxun

    2016-12-01

    Cardiac tissue engineering (CTE) has developed rapidly, but a great challenge remains in finding practical scaffold materials for the construction of engineered cardiac tissues. Carbon nanohorns (CNHs) may be a potential candidate due to their special structure and properties. The purpose of this study was to assess the effect of CNHs on the biological behavior of neonatal rat ventricular myocytes (NRVMs) for CTE applications. CNHs were incorporated into collagen to form growth substrates for NRVMs. Transmission electron microscopy (TEM) observations demonstrated that CNHs exhibited a good affinity to collagen. Moreover, it was found that CNH-embedded substrates enhanced adhesion and proliferation of NRVMs. Immunohistochemical staining, western blot analysis, and intracellular calcium transient measurements indicated that the addition of CNHs significantly increased the expression and maturation of electrical and mechanical proteins (connexin-43 and N-cadherin). Bromodeoxyuridine staining and a Cell Counting Kit-8 assay showed that CNHs have the ability to inhibit the proliferation of cardiac fibroblasts. These findings suggest that CNHs can have a valuable effect on the construction of engineered cardiac tissues and may be a promising scaffold for CTE. PMID:27263018

  19. Cell proliferation and hair cell addition in the ear of the goldfish, Carassius auratus

    Science.gov (United States)

    Lanford, P. J.; Presson, J. C.; Popper, A. N.

    1996-01-01

    Cell proliferation and hair cell addition have not been studied in the ears of otophysan fish, a group of species who have specialized hearing capabilities. In this study we used the mitotic S-phase marker bromodeoxyuridine (BrdU) to identify proliferating cells in the ear of one otophysan species, Carassius auratus (the goldfish). Animals were sacrificed at 3 h or 5 days postinjection with BrdU and processed for immunocytochemistry. The results of the study show that cell proliferation occurs in all of the otic endorgans and results in the addition of new hair cells. BrdU-labeled cells were distributed throughout all epithelia, including the primary auditory endorgan (saccule), where hair cell phenotypes vary considerably along the rostrocaudal axis. This study lays the groundwork for our transmission electron microscopy study of proliferative cells in the goldfish ear (Presson et al., Hearing Research 100 (1996) 10-20) as well as future studies of hair cell development in this species. The ability to predict, based on epithelial location, the future phenotype of developing hair cells in the saccule of the goldfish make that endorgan a particularly powerful model system for the investigation of early hair cell differentiation.

  20. Neurogenesis in an early protostome relative: progenitor cells in the ventral nerve center of chaetognath hatchlings are arranged in a highly organized geometrical pattern.

    Science.gov (United States)

    Perez, Yvan; Rieger, Verena; Martin, Elise; Müller, Carsten H G; Harzsch, Steffen

    2013-05-01

    Emerging evidence suggests that Chaetognatha represent an evolutionary lineage that is the sister group to all other Protostomia thus promoting these animals as a pivotal model for our understanding of bilaterian evolutionary history. We have analyzed the proliferation of neuronal progenitor cells in the developing ventral nerve center (VNC) of Spadella cephaloptera hatchlings. To that end, for the first time in Chaetognatha, we performed in vivo incorporation experiments with the S-phase specific mitosis marker bromodeoxyuridine (BrdU). Our experiments provide evidence for a high level of mitotic activity in the VNC for ca. 3 days after hatching. Neurogenesis is carried by presumptive neuronal progenitor cells that cycle rapidly and most likely divide asymmetrically. These progenitors are arranged in a distinct grid-like geometrical pattern including about 35 transverse rows. Considering Chaetognaths to be an early offshoot of the protostome lineage we conclude that the presence of neuronal progenitor cells with asymmetric division seems to be a feature that is rooted deeply in the Metazoa. In the light of previous evidence indicating the presence of serially iterated peptidergic neurons with individual identities in the chaetognath VNC, we discuss if these neuronal progenitor cells give rise to distinct lineages. Furthermore, we evaluate the serially iterated arrangement of the progenitor cells in the light of evolution of segmentation. PMID:23483730

  1. Temporal and spatial distribution of metabotropic glutamate receptor 5 during development in the rat cortex and hippocampus

    Institute of Scientific and Technical Information of China (English)

    Xinli Xiao; Ming Hu; Pengbo Yang; Lin Zhang; Xinlin Chen; Yong Liu

    2011-01-01

    Metabotropic glutamate receptor 5 (mGluR5) is expressed by neurons in zones of active neurogenesis and is involved in the development of neural stem cells in vivo and in vitro. We examined the expression of mGluR5 in the cortex and hippocampus of rats during various prenatal and postnatal periods using immunohistochemistry. During prenatal development, mGluR5 was primarily localized to neuronal somas in the forebrain. During early postnatal periods, the receptor was mainly present on somas in the cortex. mGluR5 immunostaining was visible in apical dendrites and in the neuropil of neurons and persisted throughout postnatal development. During this period, pyramidal neurons were strongly labeled for the receptor. In the hippocampal CA1 region, mGluR5 immunoreactivity was more intense in the stratum oriens, stratum radiatum, and lacunosum moleculare at P0, P5 and P10 relative to P60. mGluR5 expression increased significantly in the molecular layer and decreased significantly in the granule cell layer of the dentate gyrus at P5, P10 and P60 in comparison with P0. Furthermore, some mGluR5-positive cells were also bromodeoxyuridine- or NeuroD-positive in the dentate gyrus at P14. These results demonstrate that mGluR5 has a differential expression pattern in the cortex and hippocampus during early growth, suggesting a role for this receptor in the control of domain specific brain developmental events.

  2. Phenytoin enhances the phosphorylation of epidermal growth factor receptor and fibroblast growth factor receptor in the subventricular zone and promotes the proliferation of neural precursor cells and oligodendrocyte differentiation.

    Science.gov (United States)

    Galvez-Contreras, Alma Y; Gonzalez-Castaneda, Rocio E; Campos-Ordonez, Tania; Luquin, Sonia; Gonzalez-Perez, Oscar

    2016-01-01

    Phenytoin is a widely used antiepileptic drug that induces cell proliferation in several tissues, such as heart, bone, skin, oral mucosa and neural precursors. Some of these effects are mediated via fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (EGFR). These receptors are strongly expressed in the adult ventricular-subventricular zone (V-SVZ), the main neurogenic niche in the adult brain. The aim of this study was to determine the cell lineage and cell fate of V-SVZ neural progenitors expanded by phenytoin, as well as the effects of this drug on EGFR/FGFR phosphorylation. Male BALB/C mice received 10 mg/kg phenytoin by oral cannula for 30 days. We analysed the proliferation of V-SVZ neural progenitors by immunohistochemistry and western blot. Our findings indicate that phenytoin enhanced twofold the phosphorylation of EGFR and FGFR in the V-SVZ, increased the number of bromodeoxyuridine (BrdU)+/Sox2+ and BrdU+/doublecortin+ cells in the V-SVZ, and expanded the population of Olig2-expressing cells around the lateral ventricles. After phenytoin removal, a large number of BrdU+/Receptor interacting protein (RIP)+ cells were observed in the olfactory bulb. In conclusion, phenytoin enhanced the phosphorylation of FGFR and EGFR, and promoted the expression of neural precursor markers in the V-SVZ. In parallel, the number of oligodendrocytes increased significantly after phenytoin removal. PMID:26370587

  3. ERK Regulates Renal Cell Proliferation and Renal Cyst Expansion in inv Mutant Mice

    International Nuclear Information System (INIS)

    Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invΔC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invΔC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants

  4. Differential Effects of Olanzapine and Haloperidol on MK-801-induced Memory Impairment in Mice

    Science.gov (United States)

    Song, Jae Chun; Seo, Mi Kyoung; Park, Sung Woo; Lee, Jung Goo; Kim, Young Hoon

    2016-01-01

    Objective We investigated the differential effects of the antipsychotic drugs olanzapine and haloperidol on MK-801-induced memory impairment and neurogenesis in mice. Methods MK-801 (0.1 mg/kg) was administered 20 minutes prior to behavioral testing over 9 days. Beginning on the sixth day of MK-801 treatment, either olanzapine (0.05 mg/kg) or haloperidol (0.05 mg/kg) was administered 40 minutes prior to MK-801 for the final 4 days. Spatial memory performance was measured using a Morris water maze (MWM) test for 9 days (four trials/day). Immunohistochemistry with bromodeoxyuridine (BrdU) was used to identify newborn cells labeled in tissue sections from the dentate gyrus of the hippocampus. Results MK-801 administration over 9 days significantly impaired memory performance in the MWM test compared to untreated controls (p<0.05) and these deficits were blocked by treatment with olanzapine (p<0.05) but not haloperidol. The administration of MK-801 also resulted in a decrease in the number of BrdU-labeled cells in the dentate gyrus (28.6%; p<0.01), which was prevented by treatment with olanzapine (p<0.05) but not haloperidol. Conclusion These results suggest that olanzapine has a protective effect against cognitive impairments induced by MK-801 in mice via the stimulating effects of neurogenesis. PMID:27489382

  5. Health assessment of gasoline and fuel oxygenate vapors: micronucleus and sister chromatid exchange evaluations.

    Science.gov (United States)

    Schreiner, Ceinwen A; Hoffman, Gary M; Gudi, Ramadevi; Clark, Charles R

    2014-11-01

    Micronucleus and sister chromatid exchange (SCE) tests were performed for vapor condensate of baseline gasoline (BGVC), or gasoline with oxygenates, methyl tert-butyl ether (G/MTBE), ethyl tert butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), t-butyl alcohol (TBA), or ethanol (G/EtOH). Sprague Dawley rats (the same 5/sex/group for both endpoints) were exposed to 0, 2000, 10,000, or 20,000mg/m(3) of each condensate, 6h/day, 5days/week over 4weeks. Positive controls (5/sex/test) were given cyclophosphamide IP, 24h prior to sacrifice at 5mg/kg (SCE test) and 40mg/kg (micronucleus test). Blood was collected from the abdominal aorta for the SCE test and femurs removed for the micronucleus test. Blood cell cultures were treated with 5μg/ml bromodeoxyuridine (BrdU) for SCE evaluation. No significant increases in micronucleated immature erythrocytes were observed for any test material. Statistically significant increases in SCE were observed in rats given BGVC alone or in female rats given G/MTBE. G/TAME induced increased SCE in both sexes at the highest dose only. Although DNA perturbation was observed for several samples, DNA damage was not expressed as increased micronuclei in bone marrow cells. Inclusion of oxygenates in gasoline did not increase the effects of gasoline alone or produce a cytogenetic hazard. PMID:24852491

  6. Murine neocortical histogenesis is perturbed by prenatal exposure to low doses of Bisphenol A.

    Science.gov (United States)

    Nakamura, Keiko; Itoh, Kyoko; Yaoi, Takeshi; Fujiwara, Yasuhiro; Sugimoto, Tohru; Fushiki, Shinji

    2006-11-01

    Bisphenol A (BPA) has been shown to disrupt thyroid hormone function. We therefore studied whether prenatal exposure to low-doses of BPA affects the morphology and the expression of some genes related to brain development in the murine fetal neocortex. Pregnant mice were injected subcutaneously with 20 microg/kg of BPA daily from embryonic day 0 (E0). Control animals received vehicle alone. For evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (BrdU) was injected intraperitoneally into pregnant mice with various regimens and the brains were processed for immunohistochemistry. The total RNA was extracted from the embryonic telencephalon at various embryonic stages. The BrdU-labeled cells examined 1 hour after BrdU injection showed no differences between the BPA-treated and control groups (n = 10, each), which indicated that the proliferation of precursor cells was not affected. The BrdU-labeled cells, analysed 2 days after BrdU injection, were decreased in the ventricular zone of BPA-treated mice at E14.5 and E16.5, whereas they were increased in the cortical plate at E14.5 as compared with those in control mice (n = 10, each). Furthermore, the expression of Math3, Ngn2, Hes1, LICAM, and THRalpha was significantly upregulated at E14.5 in the BPA-treated group. These results suggested that BPA might disrupt normal neocortical development by accelerating neuronal differentiation/migration. PMID:16902998

  7. Interspecies cytogenetic comparisons: Studies with x-radiation and bleomycin sulfate

    International Nuclear Information System (INIS)

    A series of in vitro experiments were conducted to determine if there are innate differences in the sensitivity of peripheral blood lymphocytes (PBLs) from different mammalian species to clastogens. Mouse, rat, and human whole blood samples were exposed to either 0, 0.38, 0.75, 1.5, or 3.0 Gy x-radiation or 0, 5, 10, 20, 40, or 80 μg/ml bleomycin for 4 hr. Bromodeoxyuridine-containing cultures were initiated and the PBLs stimulated to divide with phytohemagglutinin. All cultures were harvested following a 3-hr colcemid treatment. Slides were made and differentially stained, and first-division metaphases were scored for chromosome aberrations. In the x-radiation studies human PBLs were significantly more sensitive than mouse PBLs which were in turn more sensitive than rat PBLs as measured by either the total percent aberrant cells or the number of dicentrics. Data from all three species could be fitted to a linear-quadratic model. Results with bleomycin suggest that the mouse and human PBLs are equally sensitive to the clastogenic effects of bleomycin. Both appeared to be more sensitive than the rat PBLs, but the variation between experiments was such that the results among species were not significantly different. These results indicate that there may be inherent differences in sensitivity among PBLs of mammalian species; however, more studies are needed to determine if the differences presented here hold for other agents. 39 refs., 2 figs., 4 tabs

  8. Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

    Science.gov (United States)

    Dono, R; Texido, G; Dussel, R; Ehmke, H; Zeller, R

    1998-08-01

    Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function. PMID:9687490

  9. Mapping small DNA sequences by fluorescence in situ hybridization directly on banded metaphase chromosomes

    International Nuclear Information System (INIS)

    A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photographic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as 1 kilobase, is applicable for larger probes, and will greatly facilitate mapping the vast number of probes being generated to study genetic disease and define the human genome. Human metaphase chromosomes were prepared from phytohemagglutinin-stimulated lymphocyte cultures synchronized with bromodeoxyuridine and thymidine. Probes were labeled with biotin-dUTP, and the hybridization signal was amplified by immunofluorescence. Chromosomes were stained with both propidium iodide and 4',6-diamidino-2-phenylindole (DAPI), producing R- and Q-banding patterns, respectively, allowing unambiguous chromosome and band identification while simultaneously visualizing the hybridization signal. Thirteen unique DNA segments have been localized to the long arm of chromosome 11 by using this technique, and localization of 10 additional probes by using radioactive in situ hybridization provides a comparison between the two procedures. These DNA segments have been mapped to all long-arm bands on chromosome 11 and in regions associated with neoplasias and inherited disorders

  10. Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

    Directory of Open Access Journals (Sweden)

    Folgueira M.A.A.K.

    2000-01-01

    Full Text Available A close correlation between vitamin D receptor (VDR abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA treatment, cells from the three lineages (HL-60, U937 and K562 differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

  11. Polynucleotide phosphorylase plays an important role in the generation of spontaneous mutations in Escherichia coli.

    Science.gov (United States)

    Becket, Elinne; Tse, Lawrence; Yung, Madeline; Cosico, Alexander; Miller, Jeffrey H

    2012-10-01

    Polynucleotide phosphorylase (PNP) plays a central role in RNA degradation, generating a pool of ribonucleoside diphosphates (rNDPs) that can be converted to deoxyribonucleoside diphosphates (dNDPs) by ribonucleotide reductase. We report here that spontaneous mutations resulting from replication errors, which are normally repaired by the mismatch repair (MMR) system, are sharply reduced in a PNP-deficient Escherichia coli strain. This is true for base substitution mutations that occur in the rpoB gene leading to Rif(r) and the gyrB gene leading to Nal(r) and for base substitution and frameshift mutations that occur in the lacZ gene. These results suggest that the increase in the rNDP pools generated by polynucleotide phosphorylase (PNP) degradation of RNA is responsible for the spontaneous mutations observed in an MMR-deficient background. The PNP-derived pool also appears responsible for the observed mutations in the mutT mutator background and those that occur after treatment with 5-bromodeoxyuridine, as these mutations are also drastically reduced in a PNP-deficient strain. However, mutation frequencies are not reduced in a mutY mutator background or after treatment with 2-aminopurine. These results highlight the central role in mutagenesis played by the rNDP pools (and the subsequent dNTP pools) derived from RNA degradation. PMID:22904280

  12. Treatment with the herbal medicine, naoxintong improves the protective effect of high-density lipoproteins on endothelial function in patients with type 2 diabetes

    Science.gov (United States)

    LV, PU; TONG, XUNLIANG; PENG, QING; LIU, YUANYUAN; JIN, HAIQIANG; LIU, RAN; SUN, WEI; PAN, BING; ZHENG, LEMIN; HUANG, YINING

    2016-01-01

    The protective effect of high-density lipoprotein (HDL) on endothelial function is impaired in patients with type 2 diabetes mellitus (T2DM), which may result in atherosclerotic complications. Naoxintong (NXT) is a compound preparation that includes Radix Astragali, Angelicae sinensis, Radix Paeoniae Rubra and Ligusticum wallichii. It is widely administered in China to prevent atherosclerotic complications. In the present study, NXT was administered to 69 patients with T2DM. HDLs were isolated from patient blood samples prior to and following the intervention. In vitro endothelial functions of HDL, including proliferation, migration, angiogenesis, and anti-apoptosis were investigated by bromodeoxyuridine, wound healing, Transwell and Matrigel tube formation assays on human umbilical vein endothelial cells (HUVECs). The results from the present study demonstrated that HUVECs treated with HDL isolated from diabetic patients following NXT therapy exhibited increased proliferative effects (10–27%; P<0.05), and improved migration ability (15–35%; P<0.05), anti-apoptotic function (23–34%; P<0.05) and angiogenesis (30–54%; P<0.001). Furthermore, the phosphorylation levels of Akt (26–36%; P<0.01) and extracellular signal-regulated kinase (16–80%; P<0.01) were increased following NXT therapy. The present in vitro study demonstrates that the protective effect of HDL on endothelial function is markedly impaired in diabetic patients who tend to develop atherosclerosis, and the impaired function may be partly abrogated by NXT. PMID:26781332

  13. Proliferative potential of recurrent intracranial meningiomas as evaluated by labelling indices of BUdR and Ki-67, and tumour doubling time

    International Nuclear Information System (INIS)

    This study was designed to provide the reciprocal relationship among labelling indices of 5-bromodeoxyuridine (BUdR LI), Ki-67 (Ki-LI), and tumour doubling time (Td) of recurrent meningiomas. In our series of 182 primary intracranial meningiomas, 46 cases recurred. The average of BUdR LI and Ki LI for nonrecurrent meningiomas were 0.77 ± 0.13 % and 4.71 ± 1.96 %, respectively. Recurrent meningiomas had significantly higher LIs at the first operation: BUdR LI was 3.77 ± 1.22 % and Ki LI was 14.78 ± 3.17 %. The recurrent ratio significantly increased with the degrees of each LI. And the linear regression analysis has demonstrated a significant correlation between BUdR and Ki LI. Td was calculated accurately by NIH, a computer software. Td showed a significant inverse correlation with each of the labelling indices. Consequently, BUdR, Ki LIs and Td of individual tumours correlate mutually well. Of the 46 recurrent cases, 4 received radiation after the operation. Td of the irradiated meningiomas tended to be longer than expected for their higher level of BUdR and Ki LIs before radiation therapy. Thus, it was shown that the radiation therapy delays the regrowth of meningiomas. (author)

  14. Ethanol in utero induces epithelial cell damage and altered kinetics in the developing rat intestine.

    Science.gov (United States)

    Estrada, G; Del Rio, J A; García-Valero, J; López-Tejero, M D

    1996-11-01

    The effect of prenatal ethanol exposure on the intestinal maturation of rat fetuses was investigated to understand the nutritional alterations found in the offspring of alcoholic mothers. Female Wistar rats were maintained on solid diet and 25% ethanol solution as drinking fluid during pregnancy, and non-alcoholic isocaloric pregnant mothers were used as controls. At birth, intestines from unsuckled pups were removed for study. The weight and length of the intestine decreased significantly when ethanol was present in utero. Ultrastructural evaluation of the epithelium revealed loss of contact between neighboring enterocytes and abnormal dilation of the cisternae of the Golgi apparatus in ethanol-exposed pups. Further, increased lysosome-like vesiculation and enhanced lysosomal beta-galactosidase activity was observed in these neonates. The total number of absorptive enterocytes in the epithelium was reduced by 30% in ethanol-exposed neonates as compared to controls, due to altered cell growth and death during fetal life. Ethanol in utero stimulated epithelial cell migration which compensated cell loss, as demonstrated by 5'-Bromodeoxyuridine labeling. These findings could have important implications for the assimilation of nutrients and failure to thrive in infants with fetal alcohol syndrome. PMID:9035346

  15. Carbon Nanohorns Promote Maturation of Neonatal Rat Ventricular Myocytes and Inhibit Proliferation of Cardiac Fibroblasts: a Promising Scaffold for Cardiac Tissue Engineering

    Science.gov (United States)

    Wu, Yujing; Shi, Xiaoli; Li, Yi; Tian, Lei; Bai, Rui; Wei, Yujie; Han, Dong; Liu, Huiliang; Xu, Jianxun

    2016-06-01

    Cardiac tissue engineering (CTE) has developed rapidly, but a great challenge remains in finding practical scaffold materials for the construction of engineered cardiac tissues. Carbon nanohorns (CNHs) may be a potential candidate due to their special structure and properties. The purpose of this study was to assess the effect of CNHs on the biological behavior of neonatal rat ventricular myocytes (NRVMs) for CTE applications. CNHs were incorporated into collagen to form growth substrates for NRVMs. Transmission electron microscopy (TEM) observations demonstrated that CNHs exhibited a good affinity to collagen. Moreover, it was found that CNH-embedded substrates enhanced adhesion and proliferation of NRVMs. Immunohistochemical staining, western blot analysis, and intracellular calcium transient measurements indicated that the addition of CNHs significantly increased the expression and maturation of electrical and mechanical proteins (connexin-43 and N-cadherin). Bromodeoxyuridine staining and a Cell Counting Kit-8 assay showed that CNHs have the ability to inhibit the proliferation of cardiac fibroblasts. These findings suggest that CNHs can have a valuable effect on the construction of engineered cardiac tissues and may be a promising scaffold for CTE.

  16. Acupuncture Induces the Proliferation and Differentiation of Endogenous Neural Stem Cells in Rats with Traumatic Brain Injury

    Science.gov (United States)

    Jiang, Shuting; Chen, Weihao; Zhang, Yimin; Zhang, Yujuan; Chen, Ailian; Dai, Qiufu; Lin, Shujun; Lin, Hanyu

    2016-01-01

    Purpose. To investigate whether acupuncture induced the proliferation and differentiation of endogenous neural stem cells (NSCs) in a rat model of traumatic brain injury (TBI). Methods. 104 Sprague-Dawley rats were randomly divided into normal, model, and acupuncture groups. Each group was subdivided into three-day (3 d), seven-day (7 d), and fourteen-day (14 d) groups. The rat TBI model was established using Feeney's freefall epidural impact method. The rats in the acupuncture group were treated at acupoints (Baihui, Shuigou, Fengfu, Yamen, and bilateral Hegu). The normal and model groups did not receive acupuncture. The establishment of the rat TBI model and the therapeutic effect of acupuncture were assessed using neurobehavioral scoring and hematoxylin-eosin staining. The proliferation and differentiation of NSCs in TBI rats were analyzed using immunofluorescence microscopy. Results. The levels of nestin-expressing cells and bromodeoxyuridine/glial fibrillary acidic protein- (BrdU/GFAP-) and BrdU/S100 calcium-binding protein B-positive and BrdU/microtubule-associated protein 2- and BrdU/galactocerebrosidase-positive cells were more significantly increased at various time points in the acupuncture group than in the model group (P Acupuncture induced the proliferation and differentiation of NSCs, thereby promoting neural repair in the TBI rats. PMID:27313641

  17. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    International Nuclear Information System (INIS)

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1+ or nestin+ stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU+ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU+ cells, very few are mash1+ or nestin+ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1+ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition

  18. The Air Liquid-interface, a Skin Microenvironment, Promotes Growth of Melanoma Cells, but not Their Apoptosis and Invasion, through Activation of Mitogen-activated Protein Kinase

    International Nuclear Information System (INIS)

    The air-liquid interface (ALI) is a common microenvironment of the skin, but it is unknown whether the ALI affects melanoma cell behaviors. Using a collagen gel invasion assay, immunohistochemistry, and Western blots, here we show that melanoma cell proliferation in cultures with an ALI is higher than melanoma cell proliferation in submerged cultures. Bromodeoxyuridine (BrdU) uptake, an indicator of cell proliferation, of melanoma cells at the ALI was about 3 times that of submerged cells, while ALI and submerged melanoma cells had similar levels of single-stranded DNA (a marker of apoptosis). The ALI enhanced the expression of Raf-1, MEK-1 and pERK-1/2 components of the mitogen-activated protein kinase (MAPK) cascade, in cells more than the submerged condition did. The increases in BrdU uptake and pERK-1/2 expression promoted by ALI was abolished by the MEK inhibitor, PD-98059. ALI-treated and submerged melanoma cells did not infiltrate into the collagen gel, and they showed no significant difference in the expression of the invasion- and motility-related molecules, matrix metalloproteinase-1 and -9, laminin 5, and filamin A. Our data indicate that the ALI, a skin microenvironment, accelerates the growth, but not the apoptosis or invasion, of melanoma cells through MAPK activation

  19. Results of 226Ra brachytherapy for T1, T2 tongue carcinoma and the clinical implication of ploidy and potential doubling time as prognostic factors

    International Nuclear Information System (INIS)

    Results of 226Ra brachytherapy for 47 patients with T1 and T2 tongue carcinoma were estimated and the clinical implication of ploidy and potential doubling time (Tpot) was discussed. Dose estimation was performed by using the dose distribution obtained from computerized dose calculators, considering Paterson-Parker's system. For T2 carcinoma, external beam irradiation (20-50 Gy) was carried out in the 12 patients and intensive chemotherapy was combined in the 16 patients. Ipsilateral neck dissection was performed for 3 out of the 4 T2N1 patients. Ploidy was analysed with flow cytometer and Tpot was obtained from in vitro labeling with bromodeoxyuridine and immunohistochemical stain. A five-year cause specific survival was 92% for T1 and 91% for T2, respectively. A five-year tumor control probability was 86% for T1 and 88% for T2, respectively. Combined intensive chemotherapy and external beam irradiation showed poorer treatment results than those without the modalities. ''Down-staging'' with the preceding combined modalities before 226Ra brachytherapy possibly had a risk to cause a geographical miss of needle implant. The significant role of Tpot was unclear. Ploidy of the tumors showed some predictive potential for the therapeutic results and the frequency of occult neck node metastasis. Optimization of brachytherapy based on the computerized dose estimation and the clinical application of biological predictors of tumors including ploidy should be considered for the treatment of tongue carcinoma. (author)

  20. Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration

    Science.gov (United States)

    Schall, K. A.; Holoyda, K. A.; Grant, C. N.; Levin, D. E.; Torres, E. R.; Maxwell, A.; Pollack, H. A.; Moats, R. A.; Frey, M. R.; Darehzereshki, A.; Al Alam, D.; Lien, C.

    2015-01-01

    Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. PMID:26089336

  1. Relationship of the demethylation of the DNA with the induction of the sister chromatid exchanges (SCE) In vivo

    International Nuclear Information System (INIS)

    The methylation of the DNA is an epigenetic modification that has an important paper in the regulation of the functionality of the genome of the organisms. It can be altered by demethylation processes, either natural or experimentally induced. The 5-azacytidine (Aza) is a compound that causes the demethylation of the DNA (dm-DNA), inducing with it, expression genic and increase in the frequency of the Sister Chromatid Exchange (SCE). The SCE is a genotoxicity indicator, caused by diverse mutagens and carcinogen. Since the biological meaning and the formation mechanism of this phenomenon has not been totally illustrious, the exploration of the relation between the dm-DNA and the induction of SCE, it could offer new knowledge to explain those queries. The purpose of this work was to study in cells of the mouse bone marrow In vivo, the effect of the Aza on the induction of SCE, based on two aspects: 1) dose answer and 2) the effectiveness of multiple exhibition. To six groups of three to five animals, they are administered Aza to dose of 5, 10, 15 or 20 mg/Kg of weight; in sharp or multiple form, previously to the bromodeoxyuridine supply and 24 h was sacrificed after this; 2 h after an injection with colchicine. Preparations of those metaphases were made, those which were dyed by means of a technique of fluorescence more Giemsa. It was observed that to sharp low dose, the Aza produced an increment in the frequency of SCE that although small it was proportional and statistically significant. To sharp and multiple high doses, the Aza doesn't cause additional increments of SCE, but if toxicity at cellular level and of individuals. It is concluded that a relationship exists between the dm-DNA and the induction of SCE. It is suggested that the total demethylation of the DNA causes 2 SCE/Cell in cells of the mouse bone marrow, or that the cytotoxicity prevents to evidence a bigger induction. (Author)

  2. Effects of co-administration of dietary sodium arsenite and an NADPH oxidase inhibitor on the rat bladder epithelium

    International Nuclear Information System (INIS)

    Arsenite (AsIII), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to produce intracellular ROS, therefore, we investigated the ability of apocynin (acetovanillone), an NADPH oxidase inhibitor, to inhibit the cytotoxicity and regenerative cell proliferation of arsenic in vitro and in vivo. Apocynin had similar effects in reducing the cytotoxicity of AsIII and dimethylarsinous acid (DMAIII) in rat urothelial cells in vitro. When tested at the same concentrations as apocynin, other antioxidants, such as L-ascorbate and N-acetylcysteine, did not inhibit AsIII-induced cytotoxicity but they were more effective at inhibiting DMAIII-induced cytotoxicity compared with apocynin. In vivo, female rats were treated for 3 weeks with 100 ppm AsIII. Immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that apocynin reduced oxidative stress partially induced by AsIII treatment on rat urothelium, and significantly reduced the cytotoxicity of superficial cells detected by scanning electron microscopy (SEM). However, based on the incidence of simple hyperplasia and the bromodeoxyuridine (BrdU) labeling index, apocynin did not inhibit AsIII-induced urothelial cell proliferation. These data suggest that the NADPH oxidase inhibitor, apocynin, may have the ability to partially inhibit arsenic-induced oxidative stress and cytotoxicity of the rat bladder epithelium in vitro and in vivo. However, apocynin did not inhibit the regenerative cell proliferation induced by arsenite in a short-term study.

  3. Spatial learning and neurogenesis: Effects of cessation of wheel running and survival of novel neurons by engagement in cognitive tasks.

    Science.gov (United States)

    Motta-Teixeira, Lívia Clemente; Takada, Silvia Honda; Machado-Nils, Aline Vilar; Nogueira, Maria Inês; Xavier, Gilberto Fernando

    2016-06-01

    Physical exercise stimulates cell proliferation in the adult dentate gyrus and facilitates acquisition and/or retention of hippocampal-dependent tasks. It is established that regular physical exercise improves cognitive performance. However, it is unclear for how long these benefits last after its interruption. Independent groups of rats received both free access to either unlocked (EXE Treatment) or locked (No-EXE Treatment) running wheels for 7 days, and daily injections of bromodeoxyuridine (BrdU) in the last 3 days. After a time delay period of either 1, 3, or 6 weeks without training, the animals were tested in the Morris water maze (MWM) either in a working memory task dependent on hippocampal function (MWM-HD) or in a visible platform searching task, independent on hippocampal function (MWM-NH). Data confirmed that exposure of rats to 7 days of spontaneous wheel running increases cell proliferation and neurogenesis. In contrast, neurogenesis was not accompanied by significant improvements of performance in the working memory version of the MWM. Longer time delays between the end of exercise and the beginning of cognitive training in the MWM resulted in lower cell survival; that is, the number of novel surviving mature neurons was decreased when this delay was 6 weeks as compared with when it was 1 week. In addition, data showed that while exposure to the MWM-HD working memory task substantially increased survival of novel neurons, exposure to the MWM-NH task did not, thus indicating that survival of novel dentate gyrus neurons depends on the engagement of this brain region in performance of cognitive tasks. © 2015 Wiley Periodicals, Inc. PMID:26669934

  4. Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis.

    Science.gov (United States)

    Dever, Daniel P; Adham, Zachariah O; Thompson, Bryan; Genestine, Matthieu; Cherry, Jonathan; Olschowka, John A; DiCicco-Bloom, Emanuel; Opanashuk, Lisa A

    2016-05-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 533-550, 2016. PMID:26243376

  5. Apoptosis and proliferation of oligodendrocyte progenitor cells in the irradiated rodent spinal cord

    International Nuclear Information System (INIS)

    Purpose: Oligodendrocytes undergo early apoptosis after irradiation. The aim of this study was to determine the relationship between oligodendroglial apoptosis and proliferation of oligodendrocyte progenitor cells (OPC) in the irradiated central nervous system. Methods and Materials: Adult rats and p53 transgenic mice were given single doses of 2 Gy, 8 Gy, or 22 Gy to the cervical spinal cord. Apoptosis was assessed using TUNEL (Tdt-mediated dUTP terminal nick-end labeling) staining or by examining nuclear morphology. Oligodendrocyte progenitor cells were identified with an NG2 antibody or by in situ hybridization for platelet-derived growth factor receptor α. Proliferation of OPC was assessed by in vivo bromodeoxyuridine (BrdU) labeling and subsequent immunohistochemistry. Because radiation-induced apoptosis of oligodendroglial cells is p53 dependent, p53 transgenic mice were used to study the relationship between apoptosis and cell proliferation. Results: Oligodendrocyte progenitor cells underwent apoptosis within 24 h of irradiation in the rat. That did not result in a change in OPC density at 24 h. Oligodendrocyte progenitor cell density was significantly reduced by 2-4 weeks, but showed recovery by 6 weeks after irradiation. An increase in BrdU-labeled cells was observed at 2 weeks after 8 Gy or 22 Gy, and proliferating cells in the rat spinal cord were immunoreactive for NG2. The mouse spinal cord showed a similar early cell proliferation after irradiation. No difference was observed in the proliferation response in the spinal cord of p53 -/- mice compared with wild type animals. Conclusions: Oligodendroglial cells undergo early apoptosis and OPC undergo early proliferation after ionizing radiation. However, apoptosis is not likely to be the trigger for early proliferation of OPC in the irradiated central nervous system

  6. On the mechanistic differences of benzene-induced leukemogenesis between wild type and p53 knockout mice

    International Nuclear Information System (INIS)

    Leukemia induction by benzene inhalation was first reported by Le Noire in 1887, described multiple cases of leukemia among Parisian cobblers. However, experimental induction of leukemia by benzene exposure was not succeeded for a hundred years, until Snyder et al. and our group reported it nearly 20 years ago. Nevertheless, the mechanistic background of benzene-induced leukemia was still an enigma until recently a benzene-induced peculiar cell kinetics of the stem/progenitor cells has been elucidated by our study, demonstrated a marked repeated oscillatory decrease in peripheral blood and bone marrow (BM) cellularity during and after benzene exposure, which epigenetically preceded and developed the leukemia more than a year later. We utilized the BUUV (bromodeoxyuridine + UV exposure) method to study stem/progenitor cell kinetics during and/or after benzene exposure. Using these methods, we were able to measure the labeling rate, cycling fraction of clonogenic progenitor cells, and other cell cycle parameters. The cycling fraction of stem/progenitor cells was found not to turn into an active hematopoiesis but to remain low during benzene inhalation and further we found evidence that the cycling fraction depression may be mediated in part by a slowing of stem/progenitor cell cycling perse by up-regulation of p21. The benzene induced leukemogenicity between mice carrying wild-type p53 and mice lacking p53 seem to differ from one another. In the case of p53 knockout mouse, DNA damage such as weak mutagenicity and or chromosomal damages are retained, and those damages participated in the induction of a consequent activation of proto-oncogenes and the like, which led cells to further neoplastic changes. In contrast, in the case of wild type mice, a dramatic oscillational change in the cell cycle of the stem cell compartment seems to be an important factor for mice carrying the p53 gene. (author)

  7. Usefulness of PKH fluorescent labelling to study leukemic cell proliferation with various cytostatic drugs or acetyl tetrapeptide – AcSDKP

    International Nuclear Information System (INIS)

    PKH67 labelling was compared for classical proliferation assessment (using S phase evaluation) to analyse the cell proliferation of 29 AML patients treated or not with various drugs. Among these drugs, the effect of tetrapeptide AcSDKP or AcSDKP-NH2 on AML cells, stimulated or not by cytokines, was also evaluated in order to determine (i) if AcSDKP was able to inhibit blast cell proliferation as it inhibits haematopoietic progenitors (ii) if AcSDKP-NH2 was more stable than AcSDKP with FBS. For PKH labeling, cells were suspended in Diluent C, and rapidly admixed with PKH67 solution at 20 μM PKH67. Staining was stopped by addition of FBS. A good correlation between PKH67 labelling and bromodeoxyuridine incorporation was obtained first with 6/9 patients for control cells, then for 11/17 AML patients treated with classical antileukemic drugs (among whom 4 were also treated with AcSDKP). The effect of AcSDKP was also studied on 7 patients. The discrepancy between both methods was essentially due to an accumulation of cells into different cycle phases measured by BrdUrd incorporation secondary to drug action and PKH67 labelling which measured the dynamic proliferation. This last method allows identifying resistant cells which still proliferate. AcSDKP or AcSDKP-NH2 induced a decrease of leukemic cell proliferation in 5/7 patients when cytokines were added (in order to stimulate proliferation) one day after tetrapeptide AcSDKP or AcSDKP-NH2. No effect on proliferation was noted when cytokines were added to AcSDKP-NH2. PKH67 labelling method is a powerful tool for cell proliferation assessment in patients with AML, even in cells treated by various drugs

  8. Cornu cervi pantotrichum Pharmacopuncture Solution Facilitate Hair Growth in C57BL/6 Mice

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    Seon-Yong Lee

    2016-06-01

    Full Text Available Objectives: Cornu cervi pantotrichum (CCP has been widely used in Korean and China, as an anti-fatigue, anti-aging, and tonic agent to enhance the functions of the reproductive and the immune systems. Because CCP has various growth factors that play important roles in the development of hair follicles, we examined whether CCP pharmacopuncture solution (CCPPS was capable of promoting hair growth in an animal model. Methods: One day after hair depilation, CCPPS were topically applied to the dorsal skin of C57BL/6 mice once a day for 15 days. Hair growth activity was evaluated by using macro- and microscopic observations. Dorsal skin tissues were stained with hematoxylin and eosin. Expressions of bromodeoxyuridine (BrdU, proliferating cell nuclear antigen (PCNA, and fibroblast growth factor (FGF-7 were examined by using immunohistochemical staining. A reverse transcription polymerase chain reaction (RT-PCR analysis was also conducted to measure the messenger RNA (mRNA expression of FGF-7. Results: CCPPS induced more active hair growth than normal saline. Histologic analysis showed enlargement of the dermal papilla, elongation of the hair shaft, and expansion of hair thickness in CCPPS treated mice, indicating that CCPPS effectively induced the development of anagen. CCPPS treatment markedly increased the expressions of BrdU and PCNA in the hair follicles of C57BL/6 mice. In addition, CCPPS up regulated the expression of FGF-7, which plays an important role in the development of hair follicles. Conclusion: These results reveal that CCPPS facilitates hair re-growth by proliferation of hair follicular cells and up-regulation of FGF-7 and suggest that CCPPS can potentially be applied as an alternative treatment for patients with alopecia.

  9. Chlropyrifos-methyl shows anti-androgenic activity without estrogenic activity in rats

    International Nuclear Information System (INIS)

    Chlorpyrifos-methyl (CPM), an organophosphate insecticide, widely used for grain storage and agriculture, has been suspected as endocrine disrupter by a few in vitro studies. This study was performed to investigate the (anti-) estrogenicity and (anti-) androgenicity of CPM in vivo using immature rat uterotrophic assay and rat Hershberger assay. CPM with or without 17β-estradiol were administered to 20 days old female rats to investigate its (anti-) estrogenic activity. Uterine and vaginal weight, uterine epithelial cell height were not affected by the treatment of CPM (2, 10, 50, 250 mg/kg). CPM 250 mg/kg potentiated relative vagina weight in 17β-estradiol treated immature female rats without any changing of uterine weight. Relative liver weight was increased with decrease of body weight by CPM 250 mg/kg treatment. Uterine cell proliferation tested with bromodeoxyuridine labeling index was not observed in CPM treated rats. CPM with or without testosterone propionate were administered to castrated rat of 51 days old for 10 days to investigate the (anti-)androgenic activity,. The weight of relative and absolute androgen-dependent accessory sex organs; seminal vesicle with coagulating glands (SV/CG), ventral prostate gland (VP), glans penis (GP), levator ani plus bulbocarvernosus muscle (LABC) and Cowper's gland (CG,) were unchanged by the treatment of CPM alone. While CPM induced the increase of relative adrenal gland weight, CPM 50 mg/kg decreased the weights of CV/CG, VP, CG and LABC without change of GP without changing of GP when it was treated with TP. In conclusion, CPM dose not show estrogenic and anti-estrogenic activity in immature female rats, but it represents anti-androgenic activity by inhibition of the TP-stimulated increase of the weight of accessory sex organs

  10. Mode of action of pulegone on the urinary bladder of F344 rats.

    Science.gov (United States)

    Da Rocha, Mitscheli S; Dodmane, Puttappa R; Arnold, Lora L; Pennington, Karen L; Anwar, Muhammad M; Adams, Bret R; Taylor, Sean V; Wermes, Clint; Adams, Timothy B; Cohen, Samuel M

    2012-07-01

    Essential oils from mint plants, including peppermint and pennyroyal oils, are used at low levels as flavoring agents in various foods and beverages. Pulegone is a component of these oils. In a 2-year bioassay, oral administration of pulegone slightly increased the urothelial tumor incidence in female rats. We hypothesized that its mode of action (MOA) involved urothelial cytotoxicity and increased cell proliferation, ultimately leading to tumors. Pulegone was administered by gavage at 0, 75, or 150 mg/kg body weight to female rats for 4 and 6 weeks. Fresh void urine and 18-h urine were collected for crystal and metabolite analyses. Urinary bladders were evaluated by light microscopy and scanning electron microscopy (SEM) and bromodeoxyuridine (BrdU) labeling index. Pulegone and its metabolites, piperitenone, piperitone, menthofuran, and menthone, were tested for cytotoxicity in rat (MYP3) and human (1T1) urothelial cells by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. No abnormal urinary crystals were observed by light microscopy. Urine samples (18-h) showed the presence of pulegone, piperitone, piperitenone, and menthofuran in both treated groups. By SEM, bladders from treated rats showed superficial necrosis and exfoliation. There was a significant increase in the BrdU labeling index in the high-dose group. In vitro studies indicated that pulegone and its metabolites, especially piperitenone, are excreted and concentrated in the urine at cytotoxic levels when pulegone is administered at high doses to female rats. The present study supports the hypothesis that cytotoxicity followed by regenerative cell proliferation is the MOA for pulegone-induced urothelial tumors in female rats. PMID:22499580

  11. Galvanic vestibular stimulation impairs cell proliferation and neurogenesis in the rat hippocampus but not spatial memory.

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    Zheng, Yiwen; Geddes, Lisa; Sato, Go; Stiles, Lucy; Darlington, Cynthia L; Smith, Paul F

    2014-05-01

    Galvanic vestibular stimulation (GVS) is a method of activating the peripheral vestibular system using direct current that is widely employed in clinical neurological testing. Since movement is recognized to stimulate hippocampal neurogenesis and movement is impossible without activation of the vestibular system, we speculated that activating the vestibular system in rats while minimizing movement, by delivering GVS under anesthesia, would affect hippocampal cell proliferation and neurogenesis, and spatial memory. Compared with the sham control group, the number of cells incorporating the DNA replication marker, bromodeoxyuridine (BrdU), was significantly reduced in the bilateral hippocampi in both the cathode left-anode right and cathode right-anode left stimulation groups (P ≤ 0.0001). The majority of the BrdU(+ve) cells co-expressed Ki-67, a marker for the S phase of the cell cycle, suggesting that these BrdU(+ve) cells were still in the cell cycle; however, there was no significant difference in the degree of co-labeling between the two stimulation groups. Single labeling for doublecortin (DCX), a marker of immature neurons, showed that while there was no significant difference between the different groups in the number of DCX(+ve) cells in the right dentate gryus, in the left dentate gyrus there was a significant decrease in the cathode left-anode right group compared with the sham controls (P ≤ 0.03). Nonetheless, when animals were tested in place recognition, object exploration and Morris water maze tasks, there were no significant differences between the GVS groups and the sham controls. These results suggest that GVS can have striking effects on cell proliferation and possibly neurogenesis in the hippocampus, without affecting spatial memory. PMID:24449222

  12. Contribution of phospholipase D in endothelin-1-mediated extracellular signal-regulated kinase activation and proliferation in rat uterine leiomyoma cells.

    Science.gov (United States)

    Robin, Philippe; Chouayekh, Sondes; Bole-Feysot, Christine; Leiber, Denis; Tanfin, Zahra

    2005-01-01

    Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth. PMID:15355882

  13. The exceptional stem cell system of Macrostomum lignano: Screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation

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    Eichberger Paul

    2007-03-01

    Full Text Available Abstract Background Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system. Results We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes – macvasa and macpiwi – and studied effects of hydroxyurea (HU treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories. Conclusion We show that Macrostomum lignano is a suitable organism to perform high throughput large

  14. ALDH expression characterizes G1-phase proliferating beta cells during pregnancy.

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    Lijuan Zhang

    Full Text Available High levels of aldehyde dehydrogenase (ALDH activity have been detected in various progenitor and stem cells. Thus, Aldefluor fluorescence, which represents precisely the ALDH activity, has been widely used for the identification, evaluation, and isolation of stem and progenitor cells. Recently, ALDH activity was detected in embryonic and adult mouse pancreas, specifically in adult centroacinar and terminal duct cells supposed to harbor endocrine and exocrine progenitor cells in the adult pancreas. Nevertheless, ALDH activity and aldeflour fluorescence have not been examined in beta cells. Here, we report a dynamic increase in the number of aldeflour+ beta cells during pregnancy. Interestingly, nearly all these aldeflour+ beta cells are positive for Ki-67, suggesting that they are in an active cell cycle (G1, S and M phases. To determine precisely at which phase beta cells activate ALDH activity and thus become aldeflour+, we co-stained insulin with additional proliferation markers, phosphohistone3 (PHH3, a marker for M-phase proliferating cells and Bromodeoxyuridine (BrdU, a marker for S-phase proliferating cells. Our data show little aldeflour+ beta cells that were positive for either PHH3, or BrdU, suggesting that beta cells activate ALDH and become Aldefluor+ when they enter G1-phase of active cell cycle, but may downregulate ALDH when they leave G1-phase and enter S phase. Our data thus reveal a potential change in ALDH activity of proliferating beta cells during pregnancy, which provides a novel method for isolation and analysis of proliferating beta cells. Moreover, our data also suggest that caution needs to be taken on interpretation of Aldefluor lineage-tracing data in pancreas.

  15. ALDH Expression Characterizes G1-Phase Proliferating Beta Cells during Pregnancy

    Science.gov (United States)

    Zhang, Lijuan; Wang, Lin; Liu, Xiaoliang; Zheng, Dongming; Liu, Sishi; Liu, Caixia

    2014-01-01

    High levels of aldehyde dehydrogenase (ALDH) activity have been detected in various progenitor and stem cells. Thus, Aldefluor fluorescence, which represents precisely the ALDH activity, has been widely used for the identification, evaluation, and isolation of stem and progenitor cells. Recently, ALDH activity was detected in embryonic and adult mouse pancreas, specifically in adult centroacinar and terminal duct cells supposed to harbor endocrine and exocrine progenitor cells in the adult pancreas. Nevertheless, ALDH activity and aldeflour fluorescence have not been examined in beta cells. Here, we report a dynamic increase in the number of aldeflour+ beta cells during pregnancy. Interestingly, nearly all these aldeflour+ beta cells are positive for Ki-67, suggesting that they are in an active cell cycle (G1, S and M phases). To determine precisely at which phase beta cells activate ALDH activity and thus become aldeflour+, we co-stained insulin with additional proliferation markers, phosphohistone3 (PHH3, a marker for M-phase proliferating cells) and Bromodeoxyuridine (BrdU, a marker for S-phase proliferating cells). Our data show little aldeflour+ beta cells that were positive for either PHH3, or BrdU, suggesting that beta cells activate ALDH and become Aldefluor+ when they enter G1-phase of active cell cycle, but may downregulate ALDH when they leave G1-phase and enter S phase. Our data thus reveal a potential change in ALDH activity of proliferating beta cells during pregnancy, which provides a novel method for isolation and analysis of proliferating beta cells. Moreover, our data also suggest that caution needs to be taken on interpretation of Aldefluor lineage-tracing data in pancreas. PMID:24787690

  16. Erythropoietin amplifies stroke-induced oligodendrogenesis in the rat.

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    Li Zhang

    Full Text Available BACKGROUND: Erythropoietin (EPO, a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia. METHODOLOGY AND PRINCIPAL FINDINGS: Recombinant human EPO (rhEPO at a dose of 5,000 U/kg (n = 18 or saline (n = 18 was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ, but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation. CONCLUSIONS: Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.

  17. Upregulation of CENP-H in tongue cancer correlates with poor prognosis and progression

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    Weng Gui-Xiang

    2009-06-01

    Full Text Available Abstract Background Centromere protein H (CENP-H is one of the fundamental components of the human active kinetochore. Recently, CENP-H was identified to be associated with tumorigenesis. This study was aimed to investigate the clinicopathologic significance of CENP-H in tongue cancer. Methods RT-PCR, real time RT-PCR and Western blot were used to examine the expression of CENP-H in tongue cancer cell lines and biopsies. CENP-H protein level in paraffin-embedded tongue cancer tissues were tested by immunohistochemical staining and undergone statistical analysis. CENP-H-knockdown stable cell line was established by infecting cells with a retroviral vector pSuper-retro-CENP-H-siRNA. The biological function of CENP-H was tested by MTT assay, colony formation assay, and Bromodeoxyuridine (BrdU incorporation assay. Results CENP-H expression was higher in tongue cancer cell lines and cancer tissues (T than that in normal cell and adjacent noncancerous tongue tissues (N, respectively. It was overexpressed in 55.95% (94/168 of the paraffin-embedded tongue cancer tissues, and there was a strong correlation between CENP-H expression and clinical stage, as well as T classification. CENP-H can predict the prognosis of tongue cancer patients especially those in early stage. Depletion of CENP-H can inhibit the proliferation of tongue cancer cells (Tca8113 and downregulate the expression of Survivin. Conclusion These findings suggested that CENP-H involves in the development and progression of tongue cancer. CENP-H might be a valuable prognostic indicator for tongue cancer patients within early stage.

  18. Short-term sleep deprivation stimulates hippocampal neurogenesis in rats following global cerebral ischemia/reperfusion.

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    Oumei Cheng

    Full Text Available Sleep deprivation (SD plays a complex role in central nervous system (CNS diseases. Recent studies indicate that short-term SD can affect the extent of ischemic damage. The aim of this study was to investigate whether short-term SD could stimulate hippocampal neurogenesis in a rat model of global cerebral ischemia/reperfusion (GCIR.One hundred Sprague-Dawley rats were randomly divided into Sham, GCIR and short-term SD groups based on different durations of SD; the short-term SD group was randomly divided into three subgroups: the GCIR+6hSD*3d-treated, GCIR+12hSD-treated and GCIR+12hSD*3d-treated groups. The GCIR rat model was induced via the bilateral occlusion of the common carotid arteries and hemorrhagic hypotension. The rats were sleep-deprived starting at 48 h following GCIR. A Morris water maze test was used to assess learning and memory ability; cell proliferation and differentiation were analyzed via 5-bromodeoxyuridine (BrdU and neuron-specific enolase (NSE, respectively, at 14 and 28 d; the expression of hippocampal BDNF was measured after 7 d.The different durations of short-term SD designed in our experiment exhibited improvement in cognitive function as well as increased hippocampal BDNF expression. Additionally, the short-term SD groups also showed an increased number of BrdU- and BrdU/NSE-positive cells compared with the GCIR group. Of the three short-term SD groups, the GCIR+12hSD*3d-treated group experienced the most substantial beneficial effects.Short-term SD, especially the GCIR+12hSD*3d-treated method, stimulates neurogenesis in the hippocampal dentate gyrus (DG of rats that undergo GCIR, and BDNF may be an underlying mechanism in this process.

  19. Effect of cytokine treatment on the neurogenesis process in the brain of soman-poisoned mice

    International Nuclear Information System (INIS)

    We previously described that enhanced proliferation of neural progenitors occurred in the subgranular zone (SGZ) of the dentate gyrus and in the subventricular zone (SVZ) of the mouse brain following soman poisoning. Then, a discrete number of these cells seemed to migrate and engraft into the main damaged brain regions (hippocampus; septum and amygdala) and subsequently differentiate into neurons. In the present study, the effect of a cytokine treatment on the neurogenesis process was evaluated. For this purpose, subcutaneous injection of a cocktail of 40 μg/kg epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) was administered daily to soman-poisoned mice (110 μg/kg soman and 5.0 mg/kg methyl nitrate atropine), from post-soman days 1 to 8. To label replicating neural progenitors, 200 mg/kg bromodeoxyuridine (BrdU) was injected twice a day between post-soman days 6 and 8. Mice were sacrificed on post-soman day 9 or 34. On post-soman day 9, the cytokine treatment had no effect on the proliferation of neural progenitors in the SVZ and SGZ, as assessed by BrdU immunochemistry. However, this treatment seemed to promote the migration of neural precursor cells from the proliferative areas towards damaged brain regions. Indeed, in the CA1 hippocampal layer of soman-poisoned mice, on post-soman day 34, the cytokine treatment increased the number of healthy pyramidal neurons stained by hemalun-eosin dye. The cytokine treatment also augmented the number of BrdU-labeled cells in the CA1 hippocampal layer and amygdala. Interestingly, the administration of cytokines resulted in the differentiation of BrdU-positive cells into new neurons in the CA1 hippocampal layer, whereas astrocytic differentiation was preferentially observed in the amygdala

  20. Involvement of over-expressed BMP4 in pentylenetetrazol kindling-induced cell proliferation in the dentate gyrus of adult rats

    International Nuclear Information System (INIS)

    The dentate gyrus (DG) of the hippocampus is one of a few regions in the adult mammalian brain characterized by ongoing neurogenesis. Proliferation of neural precursors in the granule cell layer of the DG has been identified in pentylenetetrazol (PTZ) kindling epilepsy model, however, little is known about the molecular mechanism. We previously reported that the expression pattern of bone morphogenetic proteins-4 (BMP4) mRNA in the hippocampus was developmentally regulated and mainly localized in the DG of the adult. To explore the role of BMP4 in epileptic activity, we detected BMP4 expression in the DG during PTZ kindling process and explore its correlation with cell proliferation combined with bromodeoxyuridine (BrdU) labeling technique. We found that dynamic changes in BMP4 level and BrdU labeled cells dependent on the kindling stage of PTZ induced seizure-prone state. The number of BMP4 mRNA-positive cells and BrdU labeled cells reached the top level 1 day after PTZ kindled, then declined to base level 2 months later. Furthermore, there was a significant correlation between increased BMP4 mRNA expression and increased number of BrdU labeled cells. After effectively blocked expression of BMP4 with antisense oligodeoxynucleotides(ASODN), the BrdU labeled cells in the dentate gyrus subgranular zone(DG-SGZ) and hilus were significantly decreased 16d after First PTZ injection and 1, 3, 7, 14d after kindled respectively. These findings suggest that increased proliferation in the DG of the hippocampus resulted from kindling epilepsy elicited by PTZ maybe be modulated by BMP4 over-expression

  1. Exercise exerts neuroprotective effects on Parkinson's disease model of rats.

    Science.gov (United States)

    Tajiri, Naoki; Yasuhara, Takao; Shingo, Tetsuro; Kondo, Akihiko; Yuan, Wenji; Kadota, Tomohito; Wang, Feifei; Baba, Tanefumi; Tayra, Judith Thomas; Morimoto, Takamasa; Jing, Meng; Kikuchi, Yoichiro; Kuramoto, Satoshi; Agari, Takashi; Miyoshi, Yasuyuki; Fujino, Hidemi; Obata, Futoshi; Takeda, Isao; Furuta, Tomohisa; Date, Isao

    2010-01-15

    Recent studies demonstrate that rehabilitation ameliorates physical and cognitive impairments of patients with stroke, spinal cord injury, and other neurological diseases and that rehabilitation also has potencies to modulate brain plasticity. Here we examined the effects of compulsive exercise on Parkinson's disease model of rats. Before 6-hydroxydopamine (6-OHDA, 20 microg) lesion into the right striatum of female SD rats, bromodeoxyuridine (BrdU) was injected to label the proliferating cells. Subsequently, at 24 h after the lesion, the rats were forced to run on the treadmill (5 days/week, 30 min/day, 11 m/min). As behavioral evaluations, cylinder test was performed at 1, 2, 3, and 4 weeks and amphetamine-induced rotational test was performed at 2 and 4 weeks with consequent euthanasia for immunohistochemical investigations. The exercise group showed better behavioral recovery in cylinder test and significant decrease in the number of amphetamine-induced rotations, compared to the non-exercise group. Correspondingly, significant preservation of tyrosine hydroxylase (TH)-positive fibers in the striatum and TH-positive neurons in the substantia nigra pars compacta (SNc) was demonstrated, compared to the non-exercise group. Additionally, the number of migrated BrdU- and Doublecortin-positive cells toward the lesioned striatum was increased in the exercise group. Furthermore, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor increased in the striatum by exercise. The results suggest that exercise exerts neuroprotective effects or enhances the neuronal differentiation in Parkinson's disease model of rats with subsequent improvement in deteriorated motor function. PMID:19900418

  2. A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor.

    Science.gov (United States)

    Fujimoto, K; Araki, K; McCarthy, D M; Sims, J R; Ren, J Q; Zhang, X; Bhide, P G

    2010-10-27

    Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence (MGE) than the lateral ganglionic eminence (LGE) or cerebral wall (CW). Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by

  3. Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: caffeine sensitive and caffeine resistant mechanism

    International Nuclear Information System (INIS)

    Replicative bypass repair of UV damage to DNA was studied in a wide variaty of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthesized after irradiation with 10 J/m2 to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionally, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimodine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative bypassing became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability

  4. κ-Opioid Receptor Expression Defines a Phenotypically Distinct Subpopulation of Astroglia: Relationship to Ca2+ Mobilization, Development, and the Antiproliferative Effect of Opioids

    Science.gov (United States)

    Gurwell, Julie A.; Duncan, Marilyn J.; Maderspach, Katalin; Stiene-Martin, Anne; Elde, Robert P.; Hauser, Kurt F.

    2016-01-01

    To assess the role of κ opioid receptors in astrocyte development, the effect of κ agonists on the growth of astroglia derived from 1–2 day-old mouse cerebra was examined in vitro. κ-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of κ receptor activation on intracellular calcium ([Ca2+]i) homeostasis and DNA synthesis. On days 6–7, as many as 50% of the astrocytes displayed κ receptor (KA8) immunoreactivity or exhibited increases in [Ca 2+]i in response to κ agonist treatment (U69,593 or U50,488H). Exposure to U69,593 (100 nM) for 72 h caused a significant reduction in number and proportion of glial fibrillary acidic protein-immunoreactive astrocytes incorporating bromodeoxyuridine (BrdU) that could be prevented by co-administering the κ antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were κ opioid receptor (KA8) immunoreactive or displayed functional increases in [Ca2+]i. Furthermore, U69,593 (100 nM) treatment failed to inhibit BrdU incorporation at 9 days in vitro. Experimental manipulations showed that κ receptor activation increases astroglial [Ca 2+]i both through influx via L-type channels and through mobilization of intracellular stores (which is an important Ca2+ signaling pathway in cell division). Collectively, these results indicate that a subpopulation of developing astrocytes express κ opioid receptors in vitro, and suggest that the activation of κ receptors mobilizes [Ca 2+]i and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing κ receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes. PMID:8930364

  5. NF-KB downregulation may be involved the depression of tumor cell proliferation mediated by human mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Ling QIAO; Tie-jun ZHAO; Feng-ze WANG; Chang-liang SHAN; Li-hong YE; Xiao-dong ZHANG

    2008-01-01

    Aim:It has been reported that stem cells are able to home to tumorigenesis and inhibit the proliferation of tumor cells.The purpose of our study was to demon-strate the molecular mechanism of the inhibitory proliferation of hepatoma cells and breast cancer cells mediated by human mesenchymal stem cells (hMSCs).Methods:The proliferation of H7402 human hepatoma cells and MCF-7 human breast cancer cells was measured by the 5-bromodeoxyuridine (BrdU) incorpora-tion assay and flow cytometry assay after the treatment with conditioned media from hMSCs culture,such as Z3 cells or BMMS-03 cells.The role of NF-kB or the phosphorylation of inhibitor kBoα (p-IkBα) in the depression of hepatoma or breast cancer cells treated with conditioned media from Z3 cells or BMMS-03 cells was examined by reporter gene assay,quantitative real-time PCR,and Western blot analysis,respectively.Results:The proliferation of H7402 cells and MCF-7 cells was decreased significantly by the BrdU incorporation assay and flow cytometry assay after treatment.The transcriptional activity and mRNA level of NF-kB were downregulated in the treated cells by reporter gene assay and quantitative real-time PCR in a dose-dependent manner.At the protein level,NF-kB and p-IkBα decreased in the treated cells by Western blot analysis.Conclusion:Conditioned media from hMSCs are able to inhibit the proliferation of tumor cells.NF-kB downregulation is one of reasons for the depression of tumor cell proliferation mediated by hMSCs.

  6. 5α-reductase inhibition suppresses testosterone-induced initial regrowth of regressed xenograft prostate tumors in animal models.

    Science.gov (United States)

    Masoodi, Khalid Z; Ramos Garcia, Raquel; Pascal, Laura E; Wang, Yujuan; Ma, Hei M; O'Malley, Katherine; Eisermann, Kurtis; Shevrin, Daniel H; Nguyen, Holly M; Vessella, Robert L; Nelson, Joel B; Parikh, Rahul A; Wang, Zhou

    2013-07-01

    Androgen deprivation therapy (ADT) is the standard treatment for patients with prostate-specific antigen progression after treatment for localized prostate cancer. An alternative to continuous ADT is intermittent ADT (IADT), which allows recovery of testosterone during off-cycles to stimulate regrowth and differentiation of the regressed prostate tumor. IADT offers patients a reduction in side effects associated with ADT, improved quality of life, and reduced cost with no difference in overall survival. Our previous studies showed that IADT coupled with 5α-reductase inhibitor (5ARI), which blocks testosterone conversion to DHT could prolong survival of animals bearing androgen-sensitive prostate tumors when off-cycle duration was fixed. To further investigate this clinically relevant observation, we measured the time course of testosterone-induced regrowth of regressed LuCaP35 and LNCaP xenograft tumors in the presence or absence of a 5ARI. 5α-Reductase inhibitors suppressed the initial regrowth of regressed prostate tumors. However, tumors resumed growth and were no longer responsive to 5α-reductase inhibition several days after testosterone replacement. This finding was substantiated by bromodeoxyuridine and Ki67 staining of LuCaP35 tumors, which showed inhibition of prostate tumor cell proliferation by 5ARI on day 2, but not day 14, after testosterone replacement. 5α-Reductase inhibitors also suppressed testosterone-stimulated proliferation of LNCaP cells precultured in androgen-free media, suggesting that blocking testosterone conversion to DHT can inhibit prostate tumor cell proliferation via an intracrine mechanism. These results suggest that short off-cycle coupled with 5α-reductase inhibition could maximize suppression of prostate tumor growth and, thus, improve potential survival benefit achieved in combination with IADT. PMID:23671262

  7. Chromosome banding and DNA replication patterns in bird karyotypes.

    Science.gov (United States)

    Schmid, M; Enderle, E; Schindler, D; Schempp, W

    1989-01-01

    The karyotypes of the domestic chicken (Gallus domesticus), Japanese quail (Coturnix coturnix), and griffon vulture (Gyps fulvus) were studied with a variety of banding techniques. The DNA replication patterns of bird chromosomes, analyzed by incorporation of 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT), are presented here for the first time. In particular, the time sequence of replication of the ZZ/ZW sex chromosomes throughout the S-phase was meticulously analyzed. BrdU and dT incorporation are very useful methods to identify homoeologies between karyotypes, as well as rearrangements that occurred in the macroautosomes during speciation. The Z chromosomes of the three birds displayed the same replication patterns, indicating a high degree of evolutionary conservation. In the homogametic male, BrdU and dT incorporation revealed no evidence of asynchronous replication between euchromatic bands in the ZZ pair. The same was true of the three Z chromosomes in a triploid-diploid chimeric chicken embryo. Minor replication asynchronies between the homologous ZZ or ZZZ chromosomes were restricted to heterochromatic C-bands. These results confirm that, in the ZZ male/ZW female sex-determining system of birds, dosage compensation for Z-linked genes does not occur by inactivation of one of the two Z chromosomes in the homogametic male. The heterochromatic W chromosomes of the three species showed bright labeling with distamycin A/mithramycin counterstain-enhanced fluorescence and exhibited significantly delayed DNA replication. The nucleolus organizers of birds, frequently located in microchromosomes, were also distinguished by bright distamycin A/mithramycin fluorescence. PMID:2630186

  8. Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1

    Science.gov (United States)

    Shibazaki, Sekiya; Yu, Zhiheng; Nishio, Saori; Tian, Xin; Thomson, R. Brent; Mitobe, Michihiro; Louvi, Angeliki; Velazquez, Heino; Ishibe, Shuta; Cantley, Lloyd G.; Igarashi, Peter; Somlo, Stefan

    2008-01-01

    Polycystic kidney disease (ADPKD) results from failure of the kidney to properly maintain three-dimensional structure after loss of either polycystin-1 or -2. Mice with kidney selective inactivation of Pkd1 during embryogenesis develop profound renal cystic disease and die from renal failure within 3 weeks of birth. In this model, cysts form exclusively from cells in which Cre recombinase is active, but the apparent pace of cyst expansion varies by segment and cell type. Intercalated cells do not participate in cyst expansion despite the presence of cilia up to at least postnatal day 21. Cystic segments show a persistent increase in proliferation as determined by bromodeoxyuridine (BrdU) incorporation; however, the absolute proliferative index is dependent on the underlying proliferative potential of kidney tubule cells. Components of the extracellular regulated kinase (MAPK/ERK) pathway from Ras through MEK1/2 and ERK1/2 to the effector P90RSK are activated in both perinatal Pkd1 and adult Pkd2 ortholgous gene disease models. The pattern of MAPK/ERK activation is focal and does not correlate with the pattern of active proliferation identified by BrdU uptake. The possibility of a causal relationship between ERK1/2 activation and cyst cell proliferation was assessed in vivo in the acute perinatal Pkd1 model of ADPKD using MEK1/2 inhibitor U0126. U0126 treatment had no effect on progression of cyst formation in this model at doses sufficient to reduce phospho-ERK1/2 in cystic kidneys. Cysts in ADPKD exhibit both increased proliferation and activation of MAPK/ERK, but cyst growth is not prevented by inhibition of ERK1/2 activation. PMID:18263604

  9. Interactions with the young down-regulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers.

    Directory of Open Access Journals (Sweden)

    Frédéric Levy

    2014-02-01

    Full Text Available New neurons are continuously added in the dentate gyrus and the olfactory bulb of mammalian brain. While numerous environmental factors controlling survival of newborn neurons have been extensively studied, regulation by social interactions is less documented. We addressed this question by investigating the influence of parturition and interactions with the young on neurogenesis in sheep mothers. Using Bromodeoxyuridine, a marker of cell division, in combination with markers of neuronal maturation, the percentage of neuroblasts and new mature neurons in the olfactory bulb and the dentate gyrus was compared between groups of parturient ewes which could interact or not with their lamb, and virgins. In addition, a morphological analysis was performed by measuring the dendritic arbor of neuroblasts in both structures. We showed that the post-partum period was associated with a decrease in olfactory and hippocampal adult neurogenesis. In the olfactory bulb, the suppressive effect on neuroblasts was dependent on interactions with the young whereas in the dentate gyrus the decrease in new mature neurons was associated with parturition. In addition, dendritic length and number of nodes of neuroblasts were significantly enhanced by interactions with the lamb in the olfactory bulb but not in the dentate gyrus. Because interactions with the young involved learning of the olfactory signature of the lamb, we hypothesize that this learning is associated with a down-regulation in olfactory neurogenesis and an enhancement of olfactory neuroblast maturation. Our assumption is that fewer new neurons decrease cell competition in the olfactory bulb and enhance maturation of those new neurons selected to participate in the learning of the young odor.

  10. In vitro expansion of the mammary stem/progenitor cell population by xanthosine treatment

    Directory of Open Access Journals (Sweden)

    Choudhary Ratan K

    2012-06-01

    Full Text Available Abstract Background Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore are of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo, and hepatic and hair follicle stem cells in vitro. In the latter, xanthosine promoted the symmetrical division of hepatic and hair follicle stem cells. The objective of this study was to determine if treating primary cultures of bovine mammary epithelial cells (MEC with xanthosine increases the stem/progenitor cell population by promoting symmetrical division of mammary stem cells. Results In vitro treatment with xanthosine increased the population of MEC during the exponential phase of cell growth, reducing the doubling time from 86 h in control cultures to 60 h in xanthosine-treated cultures. The bromodeoxyuridine (BrdU labeling index and the proportion of MEC in S-phase both were increased by xanthosine treatment, indicating that increased cell accretion was due to increased cell proliferation. Analysis of daughter-pairs indicated that xanthosine promoted a shift from asymmetric to symmetric cell division. Moreover, the 30 % increase in symmetric cell division was concomitant with an increase in the proportion of MEC that were positive for a putative stem cell marker (FNDC3B and a trend toward increased telomerase activity. These results suggest that xanthosine treatment in vitro can increase cell proliferation, promote symmetric cell division and enhance stem/progenitor cell activity. Conclusions Xanthosine treatment increased the proliferation rate of bovine MEC in vitro. This was likely to be mediated by an increase in the proportion of stem/progenitor cells in the MEC population due to promotion of symmetrical stem cell division by xanthosine.

  11. The epidermal growth factor receptor decreases Stathmin 1 and triggers catagen entry in the mouse.

    Science.gov (United States)

    Bichsel, Kyle J; Hammiller, Brianna; Trempus, Carol S; Li, Yanhua; Hansen, Laura A

    2016-04-01

    The epidermal growth factor receptor (EGFR) is necessary for normal involution of hair follicles after the growth phase of anagen, although the mechanisms through which it acts are not well understood. In this report, we used transcriptional profiling of microdissected hair follicles from mice with skin-targeted deletion of Egfr to investigate how EGFR activation triggers catagen. Immunofluorescence for phospho-EGFR in mouse skin revealed increased activation of EGFR in follicular keratinocytes at catagen onset. Consistent with other models of EGFR deficiency, mice with skin-targeted deletion of Egfr (Krt14-Cre(+) /Egfr(fl/fl) ) exhibited a delayed and asynchronous catagen entry. Transcriptional profiling at the time of normal catagen onset at post-natal day (P) 17 revealed increased expression of the mitotic regulator Rcc2 in hair follicles lacking EGFR. Rcc2 protein was strongly immunopositive in the nuclei of control follicular keratinocytes at P16 then rapidly decreased until it was undetectable between P18 and 21. In contrast, Rcc2 expression continued in Egfr mutant follicles throughout this period. Proliferation, measured by bromodeoxyuridine incorporation, was also significantly increased in Egfr mutant follicular keratinocytes compared to controls at P18-21. Similarly, Rcc2-regulated mitotic regulator Stathmin 1 was strikingly reduced in control but not Egfr mutant follicles between P17 and P19. Deletion of Stmn1, in turn, accelerated catagen entry associated with premature cessation of proliferation in the hair follicles. These data reveal EGFR suppression of mitotic regulators including Rcc2 and Stathmin 1 as a mechanism for catagen induction in mouse skin. PMID:26661905

  12. Patterns of olfactory bulb neurogenesis in the adult zebrafish are altered following reversible deafferentation.

    Science.gov (United States)

    Trimpe, Darcy M; Byrd-Jacobs, Christine A

    2016-09-01

    Adult brain plasticity can be investigated using reversible methods that remove afferent innervation but allow return of sensory input. Repeated intranasal irrigation with Triton X-100 in adult zebrafish diminishes innervation to the olfactory bulb, resulting in a number of alterations in bulb structure and function, and cessation of the treatment allows for reinnervation and recovery. Using bromodeoxyuridine, Hu, and caspase-3 immunoreactivity we examined cell proliferation, differentiation, migration, and survival under conditions of acute and chronic deafferentation and reafferentation. Cell proliferation within the olfactory bulb was not influenced by acute or chronic deafferentation or reafferentation, but cell fate (including differentiation, migration, and/or survival of newly formed cells) was affected. We found that chronic deafferentation caused a bilateral increase in the number of newly formed cells that migrated into the bulb, although the amount of cell death of these new cells was significantly increased compared to untreated fish. Reafferentation also increased the number of newly formed cells migrating into both bulbs, suggesting that the deafferentation effect on cell fate was maintained. Reafferentation resulted in a decrease in newly formed cells that became neurons and, although death of newly formed cells was not altered from control levels, survival was reduced in relation to that seen in chronically deafferented fish. The potential effect of age on cell genesis was also examined. While the amount of cell migration into the olfactory bulbs was not affected by fish age, more of the newly formed cells became neurons in older fish. Younger fish displayed more cell death under conditions of chronic deafferentation. In sum, our results show that reversible deafferentation affects several aspects of cell fate, including cell differentiation, migration, and survival, and age of the fish influences the response to deafferentation. PMID:27343831

  13. Short term morphine exposure in vitro alters proliferation and differentiation of neural progenitor cells and promotes apoptosis via mu receptors.

    Directory of Open Access Journals (Sweden)

    Dafna Willner

    Full Text Available Chronic morphine treatment inhibits neural progenitor cell (NPC progression and negatively effects hippocampal neurogenesis. However, the effect of acute opioid treatment on cell development and its influence on NPC differentiation and proliferation in vitro is unknown. We aim to investigate the effect of a single, short term exposure of morphine on the proliferation, differentiation and apoptosis of NPCs and the mechanism involved.Cell cultures from 14-day mouse embryos were exposed to different concentrations of morphine and its antagonist naloxone for 24 hours and proliferation, differentiation and apoptosis were studied. Proliferating cells were labeled with bromodeoxyuridine (BrdU and cell fate was studied with immunocytochemistry.Cells treated with morphine demonstrated decreased BrdU expression with increased morphine concentrations. Analysis of double-labeled cells showed a decrease in cells co-stained for BrdU with nestin and an increase in cells co-stained with BrdU and neuron-specific class III β-tubuline (TUJ1 in a dose dependent manner. Furthermore, a significant increase in caspase-3 activity was observed in the nestin- positive cells. Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine.Short term morphine exposure induced inhibition of NPC proliferation and increased active caspase-3 expression in a dose dependent manner. Morphine induces neuronal and glial differentiation and decreases the expression of nestin- positive cells. These effects were reversed with the addition of the opioid antagonist naloxone. Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival.

  14. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, A.T.; van Zeeland, A.A.; Zwanenburg, T.S.

    1983-01-01

    The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, the influence of 3AB on DNA repair was measured following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. The extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines has been studied, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the frequencies of spontaneously occurring chromosomal aberrations in A-T and FA cells. 3AB does not influence the frequencies of SCEs induced by UV or mitomycin C (MMC) in CHO cells. However, it increases the frequencies of SCEs induced by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Under the conditions in which 3AB increases the frequencies of spontaneously occurring as well as induced SCEs, it does not increase the frequencies of point mutations in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. 3AB does not influence the amount of repair replication following dimethylsulphate (DMS) treatment of human fibroblasts, or UV irradiated human lymphocytes.

  15. Radiation-induced changes in the kinetics of glomerular and tubular cells in the pig kidney

    International Nuclear Information System (INIS)

    Both kidneys of 13 mature female Large White pigs were irradiated with a single dose of 9.8 Gy 60Co γ rays. The pigs were killed serially between 2 to 24 weeks after irradiation. One hour prior to sacrifice bromodeoxyuridine (BrdU) (500 mg/pig) was injected intravenously. At postmortem the kidneys were removed and tissue was taken to prepare cell suspensions. The labeling index (LI) of these suspensions was determined using flow cytometry. In vivo BrdU incorporation in tubular and glomerular cells was determined immunohistochemically. The kinetics of glomerular and tubular cells was evaluated by counting the number of labeled cells/glomerules and the number of labeled tubular cells/fields of view. An average of 1200 glomeruli and 1500 fields of view/time were counted. Similar analyses were performed on renal tissue from unirradiated control animals. Flow cytometry revealed rapid and significant increases in the LI of kidney cells; 2 weeks after irradiation the LI increased from a control value of 0.18 ± 0.01 to 1.23 ± 0.22% (P < 0.001). By 4 weeks the maximal value of 2.45 ± 0.36% was seen; the LI then declined progressively but at 24 weeks after irradiation still remained significantly above control values (P < 0.001). A similar pattern of response was determined by counting the laveled glomerular and tubular cells identified immunohistochemically. However, the increase in labeled glomerular cells occurred 2 weeks after irradiation, whereas that for the tubules occurred 4 weeks after irradiation. These findings indicate that irradiation of the kidney, classically regarded as a open-quotes late-respondingclose quotes organ, is associated with rapid and significant changes in the kinetics of both tubular and glomerular cells. 28 refs., 4 figs

  16. Commonly consumed and specialty dietary mushrooms reduce cellular proliferation in MCF-7 human breast cancer cells.

    Science.gov (United States)

    Martin, Keith R; Brophy, Sara K

    2010-11-01

    Worldwide, over one million women will be newly diagnosed with breast cancer in the next year. Moreover, breast cancer is the second leading cause of cancer death in the USA. An accumulating body of evidence suggests that consumption of dietary mushrooms can protect against breast cancer. In this study, we tested and compared the ability of five commonly consumed or specialty mushrooms to modulate cell number balance in the cancer process using MCF-7 human breast cancer cells. Hot water extracts (80°C for 2 h) of maitake (MT, Grifola frondosa), crimini (CRIM, Agaricus bisporus), portabella (PORT, Agaricus bisporus), oyster (OYS, Pleurotus ostreatus) and white button (WB, Agaricus bisporus) mushrooms or water alone (5% v/v) were incubated for 24 h with MCF-7 cells. Cellular proliferation determined by bromodeoxyuridine incorporation was significantly (P mushrooms, with MT and OYS being the most effective. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction, an often used mitochondrion-dependent marker of proliferation, was unchanged although decreased (P > 0.05) by 15% with OYS extract. Lactate dehydrogenase release, as a marker of necrosis, was significantly increased after incubation with MT but not with other test mushrooms. Furthermore, MT extract significantly increased apoptosis, or programmed cell death, as determined by terminal deoxynucleotidyl end labeling method, whereas other test mushrooms displayed trends of ∼15%. The total numbers of cells per flask, determined by hemacytometry, were not different from control cultures. Overall, all test mushrooms significantly suppressed cellular proliferation, with MT further significantly inducing apoptosis and cytotoxicity in human breast cancer cells. This suggests that both common and specialty mushrooms may be chemoprotective against breast cancer. PMID:20921274

  17. Chronic hypoxia induces the activation of the Wnt/β-catenin signaling pathway and stimulates hippocampal neurogenesis in wild-type and APPswe-PS1ΔE9 transgenic mice in vivo

    Science.gov (United States)

    Varela-Nallar, Lorena; Rojas-Abalos, Macarena; Abbott, Ana C.; Moya, Esteban A.; Iturriaga, Rodrigo; Inestrosa, Nibaldo C.

    2014-01-01

    Hypoxia modulates proliferation and differentiation of cultured embryonic and adult stem cells, an effect that includes β-catenin, a key component of the canonical Wnt signaling pathway. Here we studied the effect of mild hypoxia on the activity of the Wnt/β-catenin signaling pathway in the hippocampus of adult mice in vivo. The hypoxia-inducible transcription factor-1α (HIF-1α) was analyzed as a molecular control of the physiological hypoxic response. Exposure to chronic hypoxia (10% oxygen for 6–72 h) stimulated the activation of the Wnt/β-catenin signaling pathway. Because the Wnt/β-catenin pathway is a positive modulator of adult neurogenesis, we evaluated whether chronic hypoxia was able to stimulate neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus. Results indicate that hypoxia increased cell proliferation and neurogenesis in adult wild-type mice as determined by Ki67 staining, Bromodeoxyuridine (BrdU) incorporation and double labeling with doublecortin (DCX). Chronic hypoxia also induced neurogenesis in a double transgenic APPswe-PS1ΔE9 mouse model of Alzheimer’s disease (AD), which shows decreased levels of neurogenesis in the SGZ. Our results show for the first time that exposure to hypoxia in vivo can induce the activation of the Wnt/β-catenin signaling cascade in the hippocampus, suggesting that mild hypoxia may have a therapeutic value in neurodegenerative disorders associated with altered Wnt signaling in the brain and also in pathological conditions in which hippocampal neurogenesis is impaired. PMID:24574965

  18. Dab2IP Regulates Neuronal Positioning, Rap1 Activity and Integrin Signaling in the Developing Cortex.

    Science.gov (United States)

    Qiao, Shuhong; Homayouni, Ramin

    2015-01-01

    Dab2IP (DOC-2/DAB2 interacting protein) is a GTPase-activating protein which is involved in various aspects of brain development in addition to its roles in tumor formation and apoptosis in other systems. In this study, we carefully examined the expression profile of Dab2IP and investigated its physiological role during brain development using a Dab2IP-knockdown (KD) mouse model created by retroviral insertion of a LacZ-encoding gene-trapping cassette. LacZ staining revealed that Dab2IP is expressed in the ventricular zone as well as the cortical plate and the intermediate zone. Immunohistochemical analysis showed that Dab2IP protein is localized in the leading process and proximal cytoplasmic regions of migrating neurons in the intermediate zone. Bromodeoxyuridine birth dating experiments in combination with immunohistochemical analysis using layer-specific markers showed that Dab2IP is important for proper positioning of a subset of layer II-IV neurons in the developing cortex. Notably, neuronal migration was not completely disrupted in the cerebral cortex of Dab2IP-KD mice and disruption of migration was not strictly layer specific. Previously, we found that Dab2IP regulates multipolar transition in cortical neurons. Others have shown that Rap1 regulates the transition from multipolar to bipolar morphology in migrating postmitotic neurons through N-cadherin signaling and somal translocation in the superficial layer of the cortical plate through integrin signaling. Therefore, we examined whether Rap1 and integrin signaling were affected in Dab2IP-KD brains. We found that Dab2IP-KD resulted in higher levels of activated Rap1 and integrin in the developing cortex. Taken together, our results suggest that Dab2IP plays an important role in the migration and positioning of a subpopulation of later-born (layers II-IV) neurons, likely through the regulation of Rap1 and integrin signaling. PMID:25721469

  19. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    International Nuclear Information System (INIS)

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 (14C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). 14C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA 14C content relative to a well-established 14C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA 14C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  20. Platycodin D Induces Tumor Growth Arrest by Activating FOXO3a Expression in Prostate Cancer in vitro and in vivo

    Science.gov (United States)

    Zhou, Rui; Lu, Zongliang; Liu, Kai; Guo, Jing; Liu, Jie; Zhou, Yong; Yang, Jian; Mi, Mantian; Xu, Hongxia

    2014-01-01

    Platycodin D (PD), a major saponin derived from Platycodin grandiflorum, exerted cytotoxicity against prostate cancer cell lines (PC3, DU145 and LNCaP cells) with IC50 values in the range of 11.17 to 26.13μmol/L, whereas RWPE-1cells (a non-malignant human prostate epithelial cell line) were not significantly affected. A further study in these cell lines showed that PD could potently affect cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis (determined by Annexin V-FITC flow cytometry) and cause cell cycle arrest (indicated by PI staining). After being treated with PD for 48 hours, DU145 and LNCaP cells were arrested in the G0 /G1 phase, and PC3 cells were arrested in the G2/M phase. A Western blotting analysis indicated that PD increased the expression of the FOXO3a transcription factor, decreased the expression of p-FOXO3a and MDM2 and increased the expression of FOXO-responsive genes, p21 and p27. MDM2 silencing (transiently by siRNA-MDM2) increased the PD-induced FOXO3a protein expression, while MDM2 overexpression (in cells transiently transfected with a pcDNA3-MDM2 plasmid) decreased the PD-induced expression of the FOXO3a protein. Moreover, PD dose-dependently inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. A Western blotting analysis of the excised xenograft tumors indicated that similar changes in protein expression also occurred in vivo. These results suggest that PD exhibits significant activity against prostate cancer in vitro and in vivo. The FOXO3a transcription factor appears to be involved in the activity of PD. Together, all of these findings provide a basis for the future development of this agent for human prostate cancer therapy. PMID:25431082

  1. Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading

    Science.gov (United States)

    Decker, R. S.; Cook, M. G.; Behnke-Barclay, M.; Decker, M. L.

    1995-01-01

    Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

  2. Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

    Science.gov (United States)

    Arguello, A A; Harburg, G C; Schonborn, J R; Mandyam, C D; Yamaguchi, M; Eisch, A J

    2008-11-11

    Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage. PMID:18832014

  3. Indirect radio-chemo-beta therapy: a targeted approach to increase biological efficiency of x-rays based on energy

    Science.gov (United States)

    Oktaria, Sianne; Corde, Stéphanie; Lerch, Michael L. F.; Konstantinov, Konstantin; Rosenfeld, Anatoly B.; Tehei, Moeava

    2015-10-01

    Despite the use of multimodal treatments incorporating surgery, chemotherapy and radiotherapy, local control of gliomas remains a major challenge. The potential of a new treatment approach called indirect radio-chemo-beta therapy using the synergy created by combining methotrexate (MTX) with bromodeoxyuridine (BrUdR) under optimum energy x-ray irradiation is assessed. 9L rat gliosarcoma cells pre-treated with 0.01 μM MTX and/or 10 μM BrUdR were irradiated in vitro with 50 kVp, 125 kVp, 250 kVp, 6 MV and 10 MV x-rays. The cytotoxicity was assessed using clonogenic survival as the radiobiological endpoint. The photon energy with maximum effect was determined using radiation sensitization enhancement factors at 10% clonogenic survival (SER10%). The cell cycle distribution was investigated using flow cytometric analysis with propidium iodide staining. Incorporation of BrUdR in the DNA was detected by the fluorescence of labelled anti-BrUdR antibodies. The radiation sensitization enhancement exhibits energy dependence with a maximum of 2.3 at 125 kVp for the combined drug treated cells. At this energy, the shape of the clonogenic survival curve of the pharmacological agents treated cells changes substantially. This change is interpreted as an increased lethality of the local radiation environment and is attributed to supplemented inhibition of DNA repair. Radiation induced chemo-beta therapy was demonstrated in vitro by the targeted activation of combined pharmacological agents with optimized energy tuning of x-ray beams on 9 L cells. Our results show that this is a highly effective form of chemo-radiation therapy.

  4. CCR9-CCL25 interactions promote cisplatin resistance in breast cancer cell through Akt activation in a PI3K-dependent and FAK-independent fashion

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    Lillard James W

    2011-05-01

    Full Text Available Abstract Background Chemotherapy heavily relies on apoptosis to kill breast cancer (BrCa cells. Many breast tumors respond to chemotherapy, but cells that survive this initial response gain resistance to subsequent treatments. This leads to aggressive cell variants with an enhanced ability to migrate, invade and survive at secondary sites. Metastasis and chemoresistance are responsible for most cancer-related deaths; hence, therapies designed to minimize both are greatly needed. We have recently shown that CCR9-CCL25 interactions promote BrCa cell migration and invasion, while others have shown that this axis play important role in T cell survival. In this study we have shown potential role of CCR9-CCL25 axis in breast cancer cell survival and therapeutic efficacy of cisplatin. Methods Bromodeoxyuridine (BrdU incorporation, Vybrant apoptosis and TUNEL assays were performed to ascertain the role of CCR9-CCL25 axis in cisplatin-induced apoptosis of BrCa cells. Fast Activated Cell-based ELISA (FACE assay was used to quantify In situ activation of PI3Kp85, AktSer473, GSK-3βSer9 and FKHRThr24 in breast cancer cells with or without cisplatin treatment in presence or absence of CCL25. Results CCR9-CCL25 axis provides survival advantage to BrCa cells and inhibits cisplatin-induced apoptosis in a PI3K-dependent and focal adhesion kinase (FAK-independent fashion. Furthermore, CCR9-CCL25 axis activates cell-survival signals through Akt and subsequent glycogen synthase kinase-3 beta (GSK-3β and forkhead in human rhabdomyosarcoma (FKHR inactivation. These results show that CCR9-CCL25 axis play important role in BrCa cell survival and low chemotherapeutic efficacy of cisplatin primarily through PI3K/Akt dependent fashion.

  5. Damaged DNA binding protein 2 plays a role in breast cancer cell growth.

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    Zilal Kattan

    Full Text Available The Damaged DNA binding protein 2 (DDB2, is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER-positive (MCF-7 and T47D, but not in ER-negative breast cancer (MDA-MB231 and SKBR3 or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold higher in ER-positive than in ER-negative tumor samples (P = 0.0208 from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase. These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

  6. Estradiol and GPER Activation Differentially Affect Cell Proliferation but Not GPER Expression in the Hippocampus of Adult Female Rats.

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    Paula Duarte-Guterman

    Full Text Available Estradiol increases cell proliferation in the dentate gyrus of the female rodent but it is not known whether the G protein-coupled estrogen receptor (GPER, a membrane receptor, is involved in this process, nor whether there are regional differences in estradiol's effects on cell proliferation. Thus, we investigated whether estradiol exerts its effects on cell proliferation in the dorsal and ventral dentate gyrus through GPER, using the GPER agonist, G1, and antagonist, G15. Ovariectomized adult female rats received a single injection of either: 17β-estradiol (10 μg, G1 (0.1, 5, 10 μg, G15 (40 μg, G15 and estradiol, or vehicle (oil, DMSO, or oil+DMSO. After 30 min, animals received an injection of bromodeoxyuridine (BrdU and were perfused 24 h later. Acute treatment with estradiol increased, while the GPER agonist G1 (5 μg decreased, the number of BrdU+ cells in the dentate gyrus relative to controls. The GPER antagonist, G15 increased the number of BrdU+ cells relative to control in the dorsal region and decreased the number of BrdU+ cells in the ventral region. However, G15 treatment in conjunction with estradiol partially eliminated the estradiol-induced increase in cell proliferation in the dorsal dentate gyrus. Furthermore, G1 decreased the expression of GPER in the dentate gyrus but not the CA1 and CA3 regions of the hippocampus. In summary, we found that activation of GPER decreased cell proliferation and GPER expression in the dentate gyrus of young female rats, presenting a potential and novel estrogen-independent role for this receptor in the adult hippocampus.

  7. Hepatocellular proliferation in response to agonists of peroxisome proliferator-activated receptor alpha: a role for kupffer cells?

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    Alsarra Ibrahim A

    2006-11-01

    Full Text Available Abstract Background It has been proposed that PPARα agonists stimulate Kupffer cells in rodents which in turn, release mitogenic factors leading to hepatic hyperplasia, and eventually cancer. However, Kupffer cells do not express PPARα receptors, and PPARα agonists stimulate hepatocellular proliferation in both TNFα- and TNFα receptor-null mice, casting doubt on the involvement of Kupffer cells in the mitogenic response to PPARα agonists. This study was therefore designed to investigate whether the PPARα agonist PFOA and the Kupffer cell inhibitor methylpalmitate produce opposing effects on hepatocellular proliferation and Kupffer cell activity in vivo, in a manner that would implicate these cells in the mitogenic effects of PPARα agonists. Methods Male Sprague-Dawley rats were treated intravenously via the tail vein with methylpalmitate 24 hrs prior to perfluorooctanoic acid (PFOA, and were sacrificed 24 hrs later, one hr after an intraperitoneal injection of bromodeoxyuridine (BrdU. Sera were analyzed for TNFα and IL-1β. Liver sections were stained immunohistochemically and quantified for BrdU incorporated into DNA. Results Data show that PFOA remarkably stimulated hepatocellular proliferation in the absence of significant changes in the serum levels of either TNFα or IL-1β. In addition, methylpalmitate did not alter the levels of these mitogens in PFOA-treated animals, despite the fact that it significantly blocked the hepatocellular proliferative effect of PFOA. Correlation between hepatocellular proliferation and serum levels of TNFα or IL-1β was extremely poor. Conclusion It is unlikely that mechanisms involving Kupffer cells play an eminent role in the hepatic hyperplasia, and consequently hepatocarcinogenicity attributed to PPARα agonists. This conclusion is based on the above mentioned published data and the current findings showing animals treated with PFOA alone or in combination with methylpalmitate to have similar

  8. Impact of treadmill running and sex on hippocampal neurogenesis in the mouse model of amyotrophic lateral sclerosis.

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    Xiaoxing Ma

    Full Text Available Hippocampal neurogenesis in the subgranular zone (SGZ of dentate gyrus (DG occurs throughout life and is regulated by pathological and physiological processes. The role of oxidative stress in hippocampal neurogenesis and its response to exercise or neurodegenerative diseases remains controversial. The present study was designed to investigate the impact of oxidative stress, treadmill exercise and sex on hippocampal neurogenesis in a murine model of heightened oxidative stress (G93A mice. G93A and wild type (WT mice were randomized to a treadmill running (EX or a sedentary (SED group for 1 or 4 wk. Immunohistochemistry was used to detect bromodeoxyuridine (BrdU labeled proliferating cells, surviving cells, and their phenotype, as well as for determination of oxidative stress (3-NT; 8-OHdG. BDNF and IGF1 mRNA expression was assessed by in situ hybridization. Results showed that: (1 G93A-SED mice had greater hippocampal neurogenesis, BDNF mRNA, and 3-NT, as compared to WT-SED mice. (2 Treadmill running promoted hippocampal neurogenesis and BDNF mRNA content and lowered DNA oxidative damage (8-OHdG in WT mice. (3 Male G93A mice showed significantly higher cell proliferation but a lower level of survival vs. female G93A mice. We conclude that G93A mice show higher hippocampal neurogenesis, in association with higher BDNF expression, yet running did not further enhance these phenomena in G93A mice, probably due to a 'ceiling effect' of an already heightened basal levels of hippocampal neurogenesis and BDNF expression.

  9. Ketamine Affects the Neurogenesis of the Hippocampal Dentate Gyrus in 7-Day-Old Rats.

    Science.gov (United States)

    Huang, He; Liu, Cun-Ming; Sun, Jie; Hao, Ting; Xu, Chun-Mei; Wang, Dan; Wu, Yu-Qing

    2016-08-01

    Ketamine has been reported to cause neonatal neurotoxicity via a neuronal apoptosis mechanism; however, no in vivo research has reported whether ketamine could affect postnatal neurogenesis in the hippocampal dentate gyrus (DG). A growing number of experiments suggest that postnatal hippocampal neurogenesis is the foundation of maintaining normal hippocampus function into adulthood. Therefore, this study investigated the effect of ketamine on hippocampal neurogenesis. Male Sprague-Dawley rats were divided into two groups: the control group (equal volume of normal saline), and the ketamine-anesthesia group (40 mg/kg ketamine in four injections at 1 h intervals). The S-phase marker 5-bromodeoxyuridine (BrdU) was administered after ketamine exposure to postnatal day 7 (PND-7) rats, and the neurogenesis in the hippocampal DG was assessed using single- or double-immunofluorescence staining. The expression of GFAP in the hippocampal DG was measured by western blot analysis. Spatial reference memory was tested by Morris water maze at 2 months after PND-7 rats exposed to ketamine treatment. The present results showed that neonatal ketamine exposure significantly inhibited neural stem cell (NSC) proliferation, decreased astrocytic differentiation, and markedly enhanced neuronal differentiation. The disruptive effect of ketamine on the proliferation and differentiation of NSCs lasted at least 1 week and disappeared by 2 weeks after ketamine exposure. Moreover, the migration of newborn neurons in the granule cell layer and the growth of astrocytes in the hippocampal DG were inhibited by ketamine on PND-37 and PND-44. Finally, ketamine caused a deficit in hippocampal-dependent spatial reference memory tasks at 2 months old. Our results suggested that ketamine may interfere with hippocampal neurogenesis and long-term neurocognitive function in PND-7 rats. These findings may provide a new perspective to explain the adult neurocognitive dysfunction induced by neonatal

  10. Curcumin Triggers p16-Dependent Senescence in Active Breast Cancer-Associated Fibroblasts and Suppresses Their Paracrine Procarcinogenic Effects

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    Siti-Fauziah Hendrayani

    2013-06-01

    Full Text Available Activated cancer-associated fibroblasts (CAFs or myofibroblasts not only facilitate tumor growth and spread but also affect tumor response to therapeutic agents. Therefore, it became clear that efficient therapeutic regimens should also take into account the presence of these supportive cells and inhibit their paracrine effects. To this end, we tested the effect of low concentrations of curcumin, a pharmacologically safe natural product, on patient-derived primary breast CAF cells. We have shown that curcumin treatment upregulates p16INK4A and other tumor suppressor proteins while inactivates the JAK2/STAT3 pathway. This reduced the level of alpha-smooth muscle actin (α-SMA and the migration/invasion abilities of these cells. Furthermore, curcumin suppressed the expression/secretion of stromal cell-derived factor-1 (SDF-1, interleukin-6 (IL-6, matrix metalloproteinase-2 (MMP-2, MMP-9, and transforming growth factor-β, which impeded their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Therefore, using different markers of senescence [senescence-associated β-galactosidase (SA-β-gal activity, Ki-67 and Lamin B1 levels, and bromodeoxyuridine incorporation], we have shown that curcumin markedly suppresses Lamin B1 and triggers DNA damage-independent senescence in proliferating but not quiescent breast stromal fibroblasts. Importantly, this curcumin-related senescence was p16INK4A-dependent and occurred with no associated inflammatory secretory phenotype. These results indicate the possible inactivation of cancer-associated myofibroblasts and present the first indication that curcumin can trigger DNA damage-independent and safe senescence in stromal fibroblasts.

  11. Repair of non-dimer DNA damages in ICB 2A frog cells exposed to solar-ultraviolet radiation in the UVB (290-320 nm) range

    International Nuclear Information System (INIS)

    The purpose of the research described in this dissertation was to investigate the repair and cellular consequences of non-dimer DNA damages induced by solar-UV irradiation of cultured I CR 2A (Rana pipiens) frog cells. Because this cell line is proficient in enzymatic photoreactivation, it was possible to induce a relatively pure population of non-dimer DNA photoproducts by exposure of cells to the Mylar-filtered solar-UV wavelengths produced by a fluorescent sunlamp followed by treatment with photoreactivating light. With a modification of bromodeoxyuridine photolysis assay, it was found that the solar-UV-induced non-dimer DNA damages were repaired by a short-patch repair mechanism in which less than 20 nucleotides were inserted into a repaired region. Similar results were also obtained for γ-irradiated cells. In contrast, excision repair of 254 nm-induced dimers was accomplished by a long-patch process in which an average of about 180 nucleotides were inserted into the repaired sites. A mutant cell line, DRP 36, hypersensitive to non-dimer DNA damages, was isolated from I CR 2A cells. It was found that the DRP 36 cells performed a significantly lower level of excision repair following the induction of non-dimer DNA damages. The results are consistent with the conclusion that the DRP 36 cells are deficient in the repair of at least one type of solar-UV-induced non-dimer DNA lesion. These experiments indicate that solar-UV-induced non-dimer DNA photoproducts behave more like the photoproducts of γ-rays than those of far-UV radiation, which are primarily pyrimidine dimers

  12. Human Placenta-Derived Adherent Cells Improve Cardiac Performance in Mice With Chronic Heart Failure

    Science.gov (United States)

    Chen, Hong-Jung; Chen, Chien-Hsi; Chang, Ming-Yao; Tsai, Da-Ching; Baum, Ellen Z.; Hariri, Robert

    2015-01-01

    Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 106 cells per mouse), medium (0.5 × 105 cells per mouse), or low (0.5 × 104 cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect. PMID:25673767

  13. Proinflammatory mediators stimulate neutrophil-directed angiogenesis.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Vascular endothelial growth factor (VEGF; vascular permeability factor) is one of the most potent proangiogenic cytokines, and it plays a central role in mediating the process of angiogenesis or new blood vessel formation. Neutrophils (PMNs) recently have been shown to produce VEGF. HYPOTHESIS: The acute inflammatory response is a potent stimulus for PMN-directed angiogenesis. METHODS: Neutrophils were isolated from healthy volunteers and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and anti-human Fas monoclonal antibody. Culture supernatants were assayed for VEGF using enzyme-linked immunosorbent assays. Culture supernatants from LPS- and TNF-alpha-stimulated PMNs were then added to human umbilical vein endothelial cells and human microvessel endothelial cells and assessed for endothelial cell proliferation using 5-bromodeoxyuridine labeling. Tubule formation was also assessed on MATRIGEL basement membrane matrix. Neutrophils were lysed to measure total VEGF release, and VEGF expression was detected using Western blot analysis. RESULTS: Lipopolysaccharide and TNF-alpha stimulation resulted in significantly increased release of PMN VEGF (532+\\/-49 and 484+\\/-80 pg\\/mL, respectively; for all, presented as mean +\\/- SEM) compared with control experiments (32+\\/-4 pg\\/mL). Interleukin 6 and Fas had no effect. Culture supernatants from LPS- and TNF-alpha-stimulated PMNs also resulted in significant increases (P<.005) in macrovascular and microvascular endothelial cell proliferation and tubule formation. Adding anti-human VEGF-neutralizing polyclonal antibody to stimulated PMN supernatant inhibited these effects. Total VEGF release following cell lysis and Western blot analysis suggests that the VEGF is released from an intracellular store. CONCLUSION: Activated human PMNs are directly angiogenic by releasing VEGF, and this has important implications for inflammation, capillary leak syndrome

  14. Remyelination after chronic spinal cord injury is associated with proliferation of endogenous adult progenitor cells after systemic administration of guanosine.

    Science.gov (United States)

    Jiang, Shucui; Ballerini, Patrizia; Buccella, Silvana; Giuliani, Patricia; Jiang, Cai; Huang, Xinjie; Rathbone, Michel P

    2008-03-01

    Axonal demyelination is a consistent pathological sequel to chronic brain and spinal cord injuries and disorders that slows or disrupts impulse conduction, causing further functional loss. Since oligodendroglial progenitors are present in the demyelinated areas, failure of remyelination may be due to lack of sufficient proliferation and differentiation of oligodendroglial progenitors. Guanosine stimulates proliferation and differentiation of many types of cells in vitro and exerts neuroprotective effects in the central nervous system (CNS). Five weeks after chronic traumatic spinal cord injury (SCI), when there is no ongoing recovery of function, intraperitoneal administration of guanosine daily for 2 weeks enhanced functional improvement correlated with the increase in myelination in the injured cord. Emphasis was placed on analysis of oligodendrocytes and NG2-positive (NG2+) cells, an endogenous cell population that may be involved in oligodendrocyte replacement. There was an increase in cell proliferation (measured by bromodeoxyuridine staining) that was attributable to an intensification in progenitor cells (NG2+ cells) associated with an increase in mature oligodendrocytes (determined by Rip+ staining). The numbers of astroglia increased at all test times after administration of guanosine whereas microglia only increased in the later stages (14 days). Injected guanosine and its breakdown product guanine accumulated in the spinal cords; there was more guanine than guanosine detected. We conclude that functional improvement and remyelination after systemic administration of guanosine is due to the effect of guanosine/guanine on the proliferation of adult progenitor cells and their maturation into myelin-forming cells. This raises the possibility that administration of guanosine may be useful in the treatment of spinal cord injury or demyelinating diseases such as multiple sclerosis where quiescent oligodendroglial progenitors exist in demyelinated plaques. PMID

  15. Frequencies of chromosomal aberrations and sister chromatid exchanges in the benthic worm Neanthes arenaceodentata exposed to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, F.L.; Rice, D.W. Jr., Moore, D.H.

    1984-07-01

    Traditional bioassays are unsuitable for assessing sublethal effects from ocean disposal of low-level radioactive waste because mortality and phenotypic responses are not anticipated. We compared the usefulness of chromosomal aberration and sister chromatid exchange (SCE) induction as measures of low-level radiation effects in a sediment-dwelling marine worm, Neanthes arenaceodentata. The SCEs, in contrast to chromosomal aberrations, do not alter the overall chromosome morphology and in mammalian cells appear to be a more sensitive indicator of DNA alterations caused by environmental mutagens. Newly hatched larvae were exposed to two radiation-exposure regimes of either x rays at a high dose rate of 0.7 Gy (70 rad)/min for as long as 5.5 min or to /sup 60/Co gamma rays at a low dose rate of from 4.8 x 10/sup -5/ to 1.2 x 10/sup -1/ Gy (0.0048 to 12 rad)/h for 24 h. After irradiation, the larvae were exposed to 3 x 10/sup -5/M bromodeoxyuridine (BrdUrd) for 28 h (x-ray-irradiated larvae) or for 54 h (/sup 60/Co-irradiated larvae). Larval cells were examined for the proportion of cells in first, second, and third or greater division. Frequencies of chromosomal aberrations and SCEs were determined in first and second division cells, respectively. Results from x-ray irradiation indicated that dose-related increases occur in chromosome and chromatid deletions, but a dose of equal or greater 2 Gy (equal to or greater than 200 rad) was required to observe a significant increase. Worm larvae receiving /sup 60/Co irradiation showed elevated SCE frequencies with a significant increase of 0.6 Gy (60 rad). We suggest that both SCEs and chromosomal aberrations may be useful for measuring effects on genetic material induced by radiation. 56 references, 7 figures, 9 tables.

  16. Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

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    Vucic V.

    2006-01-01

    Full Text Available Gamma-irradiation (gamma-IR is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD, specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001 antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control to 49% (IR cells, with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

  17. Ontogenesis of NADPH-diaphorase positive neurons in guinea pig neocortex

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    Chao eLiu

    2015-02-01

    Full Text Available In mammalian cerebrum there exist two distinct types of interneurons expressing nitric oxide synthase (NOS. Type I neurons are large in size and exhibit heavy nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d histochemical reaction, while type II cells are small with light NADPH-d reactivity. The time of origin of these cortical neurons relative to corticogenesis remains largely unclear among mammals. Here we explored this issue in guinea pigs using cell birth-dating and double-labeling methods. Bromodeoxyuridine (BrdU pulse-chasing (2 doses at 50 mg/kg, 12 hours apart was given to time-pregnant mothers, followed by quantification of NADPH-d/BrdU colocalization in the parietal and temporal neocortex in offspring at postnatal day 0 (P0, P30 and P60. Type I neurons were partially colabeled with BrdU at P0, P30 and P60 following pulse-chasing at embryonic day 21 (E21, E28 and E35, varied from 2% to 11.3% of total population of these neurons for the three time groups. Type II neurons were partially colabeled for BrdU following pulse-chasing at E21, E28, E35 and E42 at P0 (8.6%-16.5% of total population for individual time groups. At P60, type II neurons were found to co-express BrdU (4.8%-11.3% of total population for individual time groups following pulse-chasing at E21, E28, E35, E42, E49, E56 and E60/61. These results indicate that in guinea pigs type I neurons are generated during early corticogenesis, whereas type II cells are produced over a wide prenatal time window persisting until birth. The data also suggest that type II nitrinergic neurons may undergo a period of development/differentiation, for over one month, before being NADPH-d reactive.

  18. Learning to learn: theta oscillations predict new learning, which enhances related learning and neurogenesis.

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    Miriam S Nokia

    Full Text Available Animals in the natural world continuously encounter learning experiences of varying degrees of novelty. New neurons in the hippocampus are especially responsive to learning associations between novel events and more cells survive if a novel and challenging task is learned. One might wonder whether new neurons would be rescued from death upon each new learning experience or whether there is an internal control system that limits the number of cells that are retained as a function of learning. In this experiment, it was hypothesized that learning a task that was similar in content to one already learned previously would not increase cell survival. We further hypothesized that in situations in which the cells are rescued hippocampal theta oscillations (3-12 Hz would be involved and perhaps necessary for increasing cell survival. Both hypotheses were disproved. Adult male Sprague-Dawley rats were trained on two similar hippocampus-dependent tasks, trace and very-long delay eyeblink conditioning, while recording hippocampal local-field potentials. Cells that were generated after training on the first task were labeled with bromodeoxyuridine and quantified after training on both tasks had ceased. Spontaneous theta activity predicted performance on the first task and the conditioned stimulus induced a theta-band response early in learning the first task. As expected, performance on the first task correlated with performance on the second task. However, theta activity did not increase during training on the second task, even though more cells were present in animals that had learned. Therefore, as long as learning occurs, relatively small changes in the environment are sufficient to increase the number of surviving neurons in the adult hippocampus and they can do so in the absence of an increase in theta activity. In conclusion, these data argue against an upper limit on the number of neurons that can be rescued from death by learning.

  19. Indirect radio-chemo-beta therapy: a targeted approach to increase biological efficiency of x-rays based on energy

    International Nuclear Information System (INIS)

    Despite the use of multimodal treatments incorporating surgery, chemotherapy and radiotherapy, local control of gliomas remains a major challenge. The potential of a new treatment approach called indirect radio-chemo-beta therapy using the synergy created by combining methotrexate (MTX) with bromodeoxyuridine (BrUdR) under optimum energy x-ray irradiation is assessed. 9L rat gliosarcoma cells pre-treated with 0.01 μM MTX and/or 10 μM BrUdR were irradiated in vitro with 50 kVp, 125 kVp, 250 kVp, 6 MV and 10 MV x-rays. The cytotoxicity was assessed using clonogenic survival as the radiobiological endpoint. The photon energy with maximum effect was determined using radiation sensitization enhancement factors at 10% clonogenic survival (SER10%). The cell cycle distribution was investigated using flow cytometric analysis with propidium iodide staining. Incorporation of BrUdR in the DNA was detected by the fluorescence of labelled anti-BrUdR antibodies. The radiation sensitization enhancement exhibits energy dependence with a maximum of 2.3 at 125 kVp for the combined drug treated cells. At this energy, the shape of the clonogenic survival curve of the pharmacological agents treated cells changes substantially. This change is interpreted as an increased lethality of the local radiation environment and is attributed to supplemented inhibition of DNA repair. Radiation induced chemo-beta therapy was demonstrated in vitro by the targeted activation of combined pharmacological agents with optimized energy tuning of x-ray beams on 9 L cells. Our results show that this is a highly effective form of chemo-radiation therapy. (paper)

  20. Co-Mg-Al oxides issued of hydrotalcite precursors for total oxidation of volatile organic compounds. Identification and toxicological impact of the by-products

    International Nuclear Information System (INIS)

    Catalysts based on Co-Mg-Al, which were used for the total oxidation of toluene, were synthesized by using the hydrotalcite pathway. The calcination allowed us to obtain various mixed oxide types (i.e. Co3O4, Co2AlO4 or CoAl2O4), presenting meso-pores of about 8 nm and high specific surface areas. The solids were tested for the total oxidation of toluene and showed a total selectivity in CO2 and H2O for 100% of toluene conversion. However, studies using diffuse reflectance infrared 'operando' and GC-MS allowed us to identify intermediary by-products stemming from the catalytic oxidation of toluene: benzene and small quantities of benzaldehyde, styrene and acetophenone. In order to contribute to the improvement of the current scientific knowledge on volatile organic compound (VOC) toxicity in humans, the lung toxicity of toluene, benzene or their association was determined by using a human epithelial lung cell model (i.e. L132 cell line). VOC cytotoxicity was studied with three complementary methods: the enzymatic activity of extracellular lactate dehydrogenase (LDH), the enzymatic activity of mitochondrial dehydrogenase (mDH), and the incorporation of 5-Bromodeoxyuridine (5-BrdU). Taken together, these results showed the occurrence of adverse effects, notably reported by significant increases in LDH activity in cell culture supernatants, 24 hours after L132 cell exposure not only to toluene alone or benzene alone, but also to their association. This original approach allowed us to integrate some toxicological parameters to help the choice of new-dedicated catalysts for the oxidative conversion of VOC. (authors)

  1. Neuroprotective efficacy of a proneurogenic compound after traumatic brain injury.

    Science.gov (United States)

    Blaya, Meghan O; Bramlett, Helen M; Naidoo, Jacinth; Pieper, Andrew A; Dietrich, W Dalton

    2014-03-01

    Traumatic brain injury (TBI) is characterized by histopathological damage and long-term sensorimotor and cognitive dysfunction. Recent studies have reported the discovery of the P7C3 class of aminopropyl carbazole agents with potent neuroprotective properties for both newborn neural precursor cells in the adult hippocampus and mature neurons in other regions of the central nervous system. This study tested, for the first time, whether the highly active P7C3-A20 compound would be neuroprotective, promote hippocampal neurogenesis, and improve functional outcomes after experimental TBI. Sprague-Dawley rats subjected to moderate fluid percussion brain injury were evaluated for quantitative immunohistochemical and behavioral changes after trauma. P7C3-A20 (10 mg/kg) or vehicle was initiated intraperitoneally 30 min postsurgery and twice per day every day thereafter for 7 days. Administration of P7C3-A20 significantly reduced overall contusion volume, preserved vulnerable anti-neuronal nuclei (NeuN)-positive pericontusional cortical neurons, and improved sensorimotor function 1 week after trauma. P7C3-A20 treatment also significantly increased both bromodeoxyuridine (BrdU)- and doublecortin (DCX)-positive cells within the subgranular zone of the ipsilateral dentate gyrus 1 week after TBI. Five weeks after TBI, animals treated with P7C3-A20 showed significantly increased BrdU/NeuN double-labeled neurons and improved cognitive function in the Morris water maze, compared to TBI-control animals. These results suggest that P7C3-A20 is neuroprotective and promotes endogenous reparative strategies after TBI. We propose that the chemical scaffold represented by P7C3-A20 provides a basis for optimizing and advancing new pharmacological agents for protecting patients against the early and chronic consequences of TBI. PMID:24070637

  2. The bradykinin B2 receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines

    International Nuclear Information System (INIS)

    The vasoactive peptide bradykinin (BK) acts as a potent growth factor for normal kidney cells, but there have been few studies on the role of BK in renal cell carcinomas. In this study, we tested the hypothesis that BK also acts as a mitogen in kidney carcinomas, and explored the effects of BK in human renal carcinoma A498 cells. The presence of mRNAs for BK B1 and BK B2 receptors in A498 cells was demonstrated by reverse transcription–polymerase chain reaction. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) with a Cytosensor microphysiometer, and assessed extracellular signal-regulated kinase (ERK) activation by Western blotting. Exposure to 100 nM of BK resulted in the rapid elevation of intracellular Ca2+, caused a ≥30% increase in NHE activity, and a ≥300% increase in ERK phosphorylation. All BK signals were blocked by HOE140, a BK B2 receptor antagonist, but not by a B1 receptor antagonist. Inhibitor studies suggest that BK-induced ERK activation requires phospholipase C and protein kinase C activities, and is Ca2+/calmodulin-dependent. The amiloride analog 5-(N-methyl-N-isobutyl)-amiloride (MIA) blocked short-term NHE activation and inhibited ERK phosphorylation, suggesting that NHE is critical for ERK activation by BK. BK induced an approximately 40% increase in the proliferation of A498 cells as assessed by bromodeoxyuridine uptake. This effect was blocked by the ERK inhibitor PD98059, and was dependent on NHE activity. We conclude that BK exerts mitogenic effects in A498 cells via the BK B2 receptor activation of growth-associated NHE and ERK

  3. Thoracic rat spinal cord contusion injury induces remote spinal gliogenesis but not neurogenesis or gliogenesis in the brain.

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    Steffen Franz

    Full Text Available After spinal cord injury, transected axons fail to regenerate, yet significant, spontaneous functional improvement can be observed over time. Distinct central nervous system regions retain the capacity to generate new neurons and glia from an endogenous pool of progenitor cells and to compensate neural cell loss following certain lesions. The aim of the present study was to investigate whether endogenous cell replacement (neurogenesis or gliogenesis in the brain (subventricular zone, SVZ; corpus callosum, CC; hippocampus, HC; and motor cortex, MC or cervical spinal cord might represent a structural correlate for spontaneous locomotor recovery after a thoracic spinal cord injury. Adult Fischer 344 rats received severe contusion injuries (200 kDyn of the mid-thoracic spinal cord using an Infinite Horizon Impactor. Uninjured rats served as controls. From 4 to 14 days post-injury, both groups received injections of bromodeoxyuridine (BrdU to label dividing cells. Over the course of six weeks post-injury, spontaneous recovery of locomotor function occurred. Survival of newly generated cells was unaltered in the SVZ, HC, CC, and the MC. Neurogenesis, as determined by identification and quantification of doublecortin immunoreactive neuroblasts or BrdU/neuronal nuclear antigen double positive newly generated neurons, was not present in non-neurogenic regions (MC, CC, and cervical spinal cord and unaltered in neurogenic regions (dentate gyrus and SVZ of the brain. The lack of neuronal replacement in the brain and spinal cord after spinal cord injury precludes any relevance for spontaneous recovery of locomotor function. Gliogenesis was increased in the cervical spinal cord remote from the injury site, however, is unlikely to contribute to functional improvement.

  4. Alkoxy radical, RCH2O, as a free radical product in x-irradiated single crystals of nucleosides and nucleotides

    International Nuclear Information System (INIS)

    The RCH2O radical is formed in x-irradiated 3'-cytidylic acid (3'CMP), 5-chlorodeoxyuridine (ClUdR), 5-bromodeoxyuridine (BUdR), adenosinexHCl (ARxHCl), and deoxyadenosine monohydrate (AdRxH2O) owing to the net loss of hydrogen from O5/sub prime/ of the primary hydroxylic group. ESR measurements were made between 78 and 100 K on single crystals of 3'CMP, ClUdR, BUdR, and ARxHCl and on polycrystalline AdRxH2O. The range of principal g values is g/sub max/=2.054--2.093, g/sub int/=2.005--2.009, g/sub min/ =1.995--2.000. The range of sum of the isotropic β-hydrogen hyperfine couplings is A/sup β/1/sub iso/ +A/sup β/2/sub iso/=141--156 G. The g tensors and A/sup β//sub iso/ values are used to determine which oxygen nonbonding orbital contains the bulk of the unpaired electron density. The unpaired electron 2p orbital symmetry axis is perpendicular to a plane determined by X(H) xxxO--C. Of the two relevant hydrogen bonds in the undamaged molecule, the X--HxxxO bond remains intact whereas the hydrogen in the O--HxxxY bond is displaced. The equation A/sup β//sub iso/=B0+B2 cos2theta, with B0 =0 and B2=101 +- 13 G describes the dependency of the β-hydrogen hyperfine coupling on the torsion angle theta, between the unpaired electron symmetry axis and the C--H/sub β/ bond of the RCH2O radical. Mechanisms of formation, factors in stabilization, and reactions of decay are discussed

  5. Evidence that tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits angiogenesis by inducing vascular endothelial cell apoptosis

    International Nuclear Information System (INIS)

    Tumor necrosis factor (TNF) and its related ligands TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL) play roles in the regulation of vascular responses, but their effect on the formation of new blood vessels (angiogenesis) is unclear. Therefore, we have examined the effects of these ligands on angiogenesis modeled with primary cultures of human umbilical vein endothelial cells (HUVEC). To examine angiogenesis in the context of the central nervous system, we have also modeled cerebral angiogenesis with the human brain endothelial cell line hCMEC/D3. Parameters studied were bromodeoxyuridine (BrdU) incorporation and cell number (MTT) assay (to assess endothelial proliferation), scratch assay (migration) and networks on Matrigel (tube formation). In our hands, neither TRAIL nor FasL (1, 10, and 100 ng/ml) had an effect on parameters of angiogenesis in the HUVEC model. In hCMEC/D3 cells by contrast, TRAIL inhibited all parameters (10-100 ng/ml, 24 h). This was due to apoptosis, since its action was blocked by the pan-caspase inhibitor zVADfmk (5 x 10-5 mol/l) and TRAIL increased caspase-3 activity 1 h after application. However FasL (100 ng/ml) increased BrdU uptake without other effects. We conclude that TRAIL has different effects on in vitro angiogenesis depending on which model is used, but that FasL is generally ineffective when applied in vitro. The data suggest that TRAIL primarily influences angiogenesis by the induction of vascular endothelial apoptosis, leading to vessel regression.

  6. Atrophy, inducible satellite cell activation, and possible denervation of supraspinatus muscle in injured human rotator-cuff muscle.

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    Gigliotti, Deanna; Leiter, Jeff R S; Macek, Bryce; Davidson, Michael J; MacDonald, Peter B; Anderson, Judy E

    2015-09-15

    The high frequency of poor outcome and chronic pain after surgical repair of shoulder rotator-cuff injury (RCI) prompted this study to explore the potential to amplify muscle regeneration using nitric oxide (NO)-based treatment. After preoperative magnetic resonance imaging (MRI), biopsies of supraspinatus and ipsilateral deltoid (as a control) were collected during reparative surgery for RCI. Muscle fiber diameter, the pattern of neuromuscular junctions observed with alpha-bungarotoxin staining, and the γ:ε subunit ratio of acetylcholine receptors in Western blots were examined in tandem with experiments to determine the in vitro responsiveness of muscle satellite cells to activation (indicated by uptake of bromodeoxyuridine, BrdU) by the NO-donor drug, isosorbide dinitrate (ISDN). Consistent with MRI findings of supraspinatus atrophy (reduced occupation ratio and tangent sign), fiber diameter was lower in supraspinatus than in deltoid. ISDN induced a significant increase over baseline (up to 1.8-fold), in the proportion of BrdU+ (activated) Pax7+ satellite cells in supraspinatus, but not in deltoid, after 40 h in culture. The novel application of denervation indices revealed a trend for supraspinatus muscle to have a higher γ:ε subunit ratio than deltoid (P = 0.13); this ratio inversely with both occupancy ratio (P < 0.05) and the proportion of clusters at neuromuscular junctions (P = 0.05). Results implicate possible supraspinatus denervation in RCI and suggest NO-donor treatment has potential to promote growth in atrophic supraspinatus muscle after RCI and improve functional outcome. PMID:26135801

  7. Hard-diet feeding recovers neurogenesis in the subventricular zone and olfactory functions of mice impaired by soft-diet feeding.

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    Chizuru Utsugi

    Full Text Available The subventricular zone (SVZ generates an immense number of neurons even during adulthood. These neurons migrate to the olfactory bulb (OB and differentiate into granule cells and periglomerular cells. The information broadcast by general odorants is received by the olfactory sensory neurons and transmitted to the OB. Recent studies have shown that a reduction of mastication impairs both neurogenesis in the hippocampus and brain functions. To examine these effects, we first measured the difference in Fos-immunoreactivity (Fos-ir at the principal sensory trigeminal nucleus (Pr5, which receives intraoral touch information via the trigeminal nerve, when female adult mice ingested a hard or soft diet to explore whether soft-diet feeding could mimic impaired mastication. Ingestion of a hard diet induced greater expression of Fos-ir cells at the Pr5 than did a soft diet or no diet. Bromodeoxyuridine-immunoreactive (BrdU-ir structures in sagittal sections of the SVZ and in the OB of mice fed a soft or hard diet were studied to explore the effects of changes in mastication on newly generated neurons. After 1 month, the density of BrdU-ir cells in the SVZ and OB was lower in the soft-diet-fed mice than in the hard-diet-fed mice. The odor preferences of individual female mice to butyric acid were tested in a Y-maze apparatus. Avoidance of butyric acid was reduced by the soft-diet feeding. We then explored the effects of the hard-diet feeding on olfactory functions and neurogenesis in the SVZ of mice impaired by soft-diet feeding. At 3 months of hard-diet feeding, avoidance of butyric acid was reversed and responses to odors and neurogenesis were recovered in the SVZ. The present results suggest that feeding with a hard diet improves neurogenesis in the SVZ, which in turn enhances olfactory function at the OB.

  8. Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APPswe/PS1ΔE9 transgenic mouse model of Alzheimer's disease

    International Nuclear Information System (INIS)

    In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APPswe/PS1ΔE9 mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APPswe/PS1ΔE9 transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.

  9. Intranasal delivery of transforming growth factor-beta1 in mice after stroke reduces infarct volume and increases neurogenesis in the subventricular zone

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    Xu Gelin

    2008-12-01

    Full Text Available Abstract Background The effect of neurotrophic factors in enhancing stroke-induced neurogenesis in the adult subventricular zone (SVZ is limited by their poor blood-brain barrier (BBB permeability. Intranasal administration is a noninvasive and valid method for delivery of neuropeptides into the brain, to bypass the BBB. We investigated the effect of treatment with intranasal transforming growth factor-β1 (TGF-β1 on neurogenesis in the adult mouse SVZ following focal ischemia. The modified Neurological Severity Scores (NSS test was used to evaluate neurological function, and infarct volumes were determined from hematoxylin-stained sections. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL labeling was performed at 7 days after middle cerebral artery occlusion (MCAO. Immunohistochemistry was used to detect bromodeoxyuridine (BrdU and neuron- or glia-specific markers for identifying neurogenesis in the SVZ at 7, 14, 21, 28 days after MCAO. Results Intranasal treatment of TGF-β1 shows significant improvement in neurological function and reduction of infarct volume compared with control animals. TGF-β1 treated mice had significantly less TUNEL-positive cells in the ipsilateral striatum than that in control groups. The number of BrdU-incorporated cells in the SVZ and striatum was significantly increased in the TGF-β1 treated group compared with control animals at each time point. In addition, numbers of BrdU- labeled cells coexpressed with the migrating neuroblast marker doublecortin (DCX and the mature neuronal marker neuronal nuclei (NeuN were significantly increased after intranasal delivery of TGF-β1, while only a few BrdU labeled cells co-stained with glial fibrillary acidic protein (GFAP. Conclusion Intranasal administration of TGF-β1 reduces infarct volume, improves functional recovery and enhances neurogenesis in mice after stroke. Intranasal TGF-β1 may have therapeutic potential for cerebrovascular

  10. Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity

    International Nuclear Information System (INIS)

    The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity

  11. Frequencies of chromosomal aberrations and sister chromatid exchanges in the benthic worm Neanthes arenaceodentata exposed to ionizing radiation

    International Nuclear Information System (INIS)

    Traditional bioassays are unsuitable for assessing sublethal effects from ocean disposal of low-level radioactive waste because mortality and phenotypic responses are not anticipated. We compared the usefulness of chromosomal aberration and sister chromatid exchange (SCE) induction as measures of low-level radiation effects in a sediment-dwelling marine worm, Neanthes arenaceodentata. The SCEs, in contrast to chromosomal aberrations, do not alter the overall chromosome morphology and in mammalian cells appear to be a more sensitive indicator of DNA alterations caused by environmental mutagens. Newly hatched larvae were exposed to two radiation-exposure regimes of either x rays at a high dose rate of 0.7 Gy (70 rad)/min for as long as 5.5 min or to 60Co gamma rays at a low dose rate of from 4.8 x 10-5 to 1.2 x 10-1 Gy (0.0048 to 12 rad)/h for 24 h. After irradiation, the larvae were exposed to 3 x 10-5M bromodeoxyuridine (BrdUrd) for 28 h (x-ray-irradiated larvae) or for 54 h (60Co-irradiated larvae). Larval cells were examined for the proportion of cells in first, second, and third or greater division. Frequencies of chromosomal aberrations and SCEs were determined in first and second division cells, respectively. Results from x-ray irradiation indicated that dose-related increases occur in chromosome and chromatid deletions, but a dose of equal or greater 2 Gy (equal to or greater than 200 rad) was required to observe a significant increase. Worm larvae receiving 60Co irradiation showed elevated SCE frequencies with a significant increase of 0.6 Gy (60 rad). We suggest that both SCEs and chromosomal aberrations may be useful for measuring effects on genetic material induced by radiation. 56 references, 7 figures, 9 tables

  12. Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: role of Aquaporin-2.

    Science.gov (United States)

    Rivarola, Valeria; Flamenco, Pilar; Melamud, Luciana; Galizia, Luciano; Ford, Paula; Capurro, Claudia

    2010-08-01

    Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes. PMID:20432437

  13. Sex and age differences in brain-derived neurotrophic factor and vimentin in the zebra finch song system: Relationships to newly generated cells.

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    Tang, Yu Ping; Wade, Juli

    2016-04-01

    The neural song circuit is enhanced in male compared with female zebra finches due to differential rates of incorporation and survival of cells between the sexes. Two double-label immunohistochemical experiments were conducted to increase the understanding of relationships between newly generated cells (marked with bromodeoxyuridine [BrdU]) and those expressing brain-derived neurotrophic factor (BDNF) and vimentin, a marker for radial glia. The song systems of males and females were investigated at posthatching day 25 during a heightened period of sexual differentiation (following BrdU injections on days 6-10) and in adulthood (following a parallel injection paradigm). In both HVC (proper name) and the robust nucleus of the arcopallium (RA), about half of the BrdU-positive cells expressed BDNF across sexes and ages. Less than 10% of the BDNF-positive cells expressed BrdU, but this percentage was greater in juveniles than adults. Across both brain regions, more BDNF-positive cells were detected in males compared with females. In RA, the number of these cells was also greater in juveniles than adults. In HVC, the average cross-sectional area covered by the vimentin labeling was greater in males than females and in juveniles compared with adults. In RA, more vimentin was detected in juveniles than adults, and within adults it was greater in females. In juveniles only, BrdU-positive cells appeared in contact with vimentin-labeled fibers in HVC, RA, and Area X. Collectively, the results are consistent with roles of BDNF- and vimentin-labeled cells influencing sexually differentiated plasticity of the song circuit. PMID:26355496

  14. Exploiting the dynamics of S-phase tracers in developing brain: interkinetic nuclear migration for cells entering versus leaving the S-phase

    Science.gov (United States)

    Hayes, N. L.; Nowakowski, R. S.

    2000-01-01

    Two S-phase markers for in vivo studies of cell proliferation in the developing central nervous system, tritiated thymidine ((3)H-TdR) and bromodeoxyuridine (BUdR), were compared using double-labeling techniques in the developing mouse cortex at embryonic day 14 (E14). The labeling efficiencies and detectability of the two tracers were approximately equivalent, and there was no evidence of significant tracer interactions that depend on order of administration. For both tracers, the loading time needed to label an S-phase cell to detectability is estimated at <0.2 h shortly after the injection of the label, but, as the concentration of the label falls, it increases to approximately 0.65 h after about 30 min. Thereafter, cells that enter the S-phase continue to become detectably labeled for approximately 5-6 h. The approximate equivalence of these two tracers was exploited to observe directly the numbers and positions of nuclei entering (labeled with the second tracer only) and leaving (labeled with the first tracer only) the S-phase. As expected, the numbers of nuclei entering and leaving the S-phase both increased as the interval between the two injections lengthened. Also, nuclei leaving the S-phase rapidly move towards the ventricular surface during G2, but, unexpectedly, the distribution of the entering nuclei does not differ significantly from the distribution of the nuclei in the S-phase. This indicates that: (1) the extent and rate of abventricular nuclear movement during G1 is variable, such that not all nuclei traverse the entire width of the ventricular zone, and (2) interkinetic nuclear movements are minimal during S-phase. Copyright 2000 S. Karger AG, Basel.

  15. Polysaccharopeptides derived from Coriolus versicolor potentiate the S-phase specific cytotoxicity of Camptothecin (CPT on human leukemia HL-60 cells

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    Jiang Pingping

    2010-04-01

    Full Text Available Abstract Background Polysaccharopeptide (PSP from Coriolus versicolor (Yunzhi is used as a supplementary cancer treatment in Asia. The present study aims to investigate whether PSP pre-treatment can increase the response of the human leukemia HL-60 cells to apoptosis induction by Camptothecin (CPT. Methods We used bivariate bromodeoxyuridine/propidium iodide (BrdUrd/PI flow cytometry analysis to measure the relative movement (RM of the BrdUrd positively labeled cells and DNA synthesis time (Ts on the HL-60 cell line. We used annexin V/PI flow cytometry analysis to quantify the viable, necrotic and apoptotic cells. The expression of cyclin E and cyclin B1 was determined with annexin V/PI flow cytometry and western blotting. Human peripheral blood mononuclear cells were used to test the cytotoxicity of PSP and CPT. Results PSP reduced cellular proliferation; inhibited cells progression through both S and G2 phase, reduced 3H-thymidine uptake and prolonged DNA synthesis time (Ts in HL-60 cells. PSP-pretreated cells enhanced the cytotoxicity of CPT. The sensitivity of cells to the cytotoxic effects of CPT was seen to be the highest in the S-phase and to a small extent of the G2 phase of the cell cycle. On the other hand, no cell death (measured by annexin V/PI was evident with the normal human peripheral blood mononuclear cells with treatment of either PSP or CPT. Conclusion The present study shows that PSP increases the sensitization of the HL-60 cells to undergo effective apoptotic cell death induced by CPT. The pattern of sensitivity of cancer cells is similar to that of HL-60 cells. PSP rapidly arrests and/or kills cells in S-phase and did not interfere with the anticancer action of CPT. PSP is a potential adjuvant to treat human leukemia as rapidly proliferating tumors is characterized by a high proportion of S-phase cells.

  16. Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris.

    Science.gov (United States)

    Walsh, Cathy J; Luer, Carl A; Noyes, David R

    2005-02-10

    The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected each year by exposure to cold weather or harmful algal blooms (red tide; Karenia brevis). Exposures can be sublethal, resulting in stressed animals that are rescued and taken to authorized facilities for rehabilitation, or lethal if exposures are prolonged or unusually severe. To investigate whether sublethal environmental exposures can impair immune function in manatees, rendering animals vulnerable to disease or death, mitogen-induced proliferation was assessed in lymphocytes from manatees exposed to cold temperatures (N=20) or red tide (N=19) in the wild, and compared to lymphocyte responses from healthy free-ranging manatees (N=32). All animals sampled for this study were adults. Lymphocytes were stimulated in vitro with either concanavalin A (ConA) or phytohemagglutinin (PHA) and proliferation was assessed after 96 h using incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into newly synthesized DNA. Proliferation of lymphocytes from manatees rescued from exposure to red tide or cold-stress was approximately one-third that of lymphocytes from healthy free-ranging manatees. To examine the direct effects of red tide toxins on lymphocyte function, mitogen-induced proliferation was assessed following co-culture of lymphocytes with K. brevis toxin extracts. Stimulation indices decreased with increasing toxin concentration, with a significant decrease in proliferation occurring in the presence of 400 ng red tide toxins/ml. When lymphocytes from cold-stressed manatees were co-cultured with red tide toxin extracts, proliferative responses were reduced even further, suggesting multiple stressors may have synergistic effects on immune function in manatees. PMID:15621310

  17. Multi-session transcranial direct current stimulation (tDCS elicits inflammatory and regenerative processes in the rat brain.

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    Maria Adele Rueger

    Full Text Available Transcranial direct current stimulation (tDCS is increasingly being used in human studies as an adjuvant tool to promote recovery of function after stroke. However, its neurobiological effects are still largely unknown. Electric fields are known to influence the migration of various cell types in vitro, but effects in vivo remain to be shown. Hypothesizing that tDCS might elicit the recruitment of cells to the cortex, we here studied the effects of tDCS in the rat brain in vivo. Adult Wistar rats (n = 16 were randomized to either anodal or cathodal stimulation for either 5 or 10 consecutive days (500 µA, 15 min. Bromodeoxyuridine (BrdU was given systemically to label dividing cells throughout the experiment. Immunohistochemical analyses ex vivo included stainings for activated microglia and endogenous neural stem cells (NSC. Multi-session tDCS with the chosen parameters did not cause a cortical lesion. An innate immune response with early upregulation of Iba1-positive activated microglia occurred after both cathodal and anodal tDCS. The involvement of adaptive immunity as assessed by ICAM1-immunoreactivity was less pronounced. Most interestingly, only cathodal tDCS increased the number of endogenous NSC in the stimulated cortex. After 10 days of cathodal stimulation, proliferating NSC increased by ∼60%, with a significant effect of both polarity and number of tDCS sessions on the recruitment of NSC. We demonstrate a pro-inflammatory effect of both cathodal and anodal tDCS, and a polarity-specific migratory effect on endogenous NSC in vivo. Our data suggest that tDCS in human stroke patients might also elicit NSC activation and modulate neuroinflammation.

  18. Glucocorticoid Suppresses Connexin 43 Expression by Inhibiting the Akt/mTOR Signaling Pathway in Osteoblasts.

    Science.gov (United States)

    Shen, Chen; Kim, Mi Ran; Noh, Jeong Mi; Kim, Su Jin; Ka, Sun-O; Kim, Ji Hye; Park, Byung-Hyun; Park, Ji Hyun

    2016-07-01

    The inhibition of proliferation or functional alteration of osteoblasts by glucocorticoids (GCs) has been recognized as an important etiology of GC-induced osteoporosis (GIO). Connexin 43 (Cx43) is the most abundant connexin isoform in bone cells and plays important roles in bone remodeling. Despite the important role of Cx43 in bone homeostasis and the prevalence of GIO, the direct action of GCs on Cx43 expression in osteoblasts has been poorly described. The aim of the present study was to evaluate how GCs affect Cx43 expression in osteoblasts. Dexamethasone (Dex) treatment decreased expression of Cx43 RNA and protein in MC3T3-E1 mouse osteoblastic cells. Reduction of Cx43 expression by Dex was dependent on the glucocorticoid receptor (GR), as it was abolished by pretreatment with a GR blocker. Treatment with PTH (1-34), a medication used for GIO management, counteracted the suppression of Cx43 by Dex. Akt or mTOR signaling modulators revealed the involvement of the Akt/mTOR signaling pathway in Dex-induced reduction of Cx43 expression. Moreover, overexpression of Cx43 significantly attenuated Dex-inhibited cell viability and proliferation, as evidenced by MTT and bromodeoxyuridine (BrdU) incorporation assay of MC3T3-E1 cells. To account for possible species or cell type differences, human primary osteoblasts were treated with Dex and similar downregulation of Cx43 by Dex was observed. In addition, immunofluorescent staining for Cx43 further demonstrated an apparent decrease in Dex-treated human osteoblasts, while analysis of lucifer yellow propagation revealed reduced gap junction intercellular communication by Dex. Collectively, these findings indicate that GCs suppress Cx43 expression in osteoblasts via GR and the Akt/mTOR signaling pathway and overexpression of Cx43 may, at least in part, rescue osteoblasts from GC-induced reductions in proliferation. PMID:26914606

  19. Radiation effects on S-phase duration, labelling index, potential doubling time and DNA distribution in head and neck cancer xenografts

    International Nuclear Information System (INIS)

    The effect of irradiation on S-phase duration (Ts), labelling index (LI), potential doubling time (Tpot), and cell cycle phase distributions was determined by DNA flow cytometry in xenografted human squamous cell carcinoma of the head and neck (SCCHN). Tumours were treated with a single dose of 3 Gy, and excised at intervals over a 90-h period. Six hours before each excision the tumours were labelled in vivo with bromodeoxyuridine (BrdUrd). Although the growth rate of irradiated tumours was comparable with that of untreated controls, analysis of BrdUrd uptake revealed a transient reduction of LI and a prolongation of Ts in irradiated tumours. Maximum mean Tpot was 931 days in irradiated tumours as compared to 13 days in untreated controls. The variations in Ts, LI and Tpot all occurred within the first hours after irradiation; during the remainder of the observation time, the values of the variables did not differ from those of untreated controls. In irradiated tumours the distribution of cells according to DNA content changed significantly on three occasions during the observation period: 1. Parallel to the initial lowering of LI and prolongation of Ts there was a transient increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S and G2; 2. at 18 h, the most pronounced cell cycle phase redistribution occurred when the G0/G1 fraction decreased and the S and G2 phase fractions increased; 3. at 66 h (i.e., approximately one cell cycle later), the pattern was the same as that after 18 h. The findings suggest that the transient prolongation of DNA replication seen in SCCHN cells immediately after a single radiation dose is a symptom of DNA damage inflicted during late G1 or early S-phase, and that this disturbance in DNA synthesis is associated with the subsequent accumulation of cells in G2 phase. (orig.)

  20. Modulation of pancreatic MIN6 insulin secretion and proliferation and extrapancreatic glucose absorption with Achillea santolina, Eryngium creticum and Pistacia atlantica extracts: in vitro evaluation

    Directory of Open Access Journals (Sweden)

    Lara Majdalawi

    2012-06-01

    Full Text Available Objective: The present in vitro studies aimed to investigate the pancreatic and extrapancreatic effects of crude aqueous extracts (AE of Achillea santolina L, Eryngium creticum Lam, and Pistacia atlantica Desf utilized in Jordan diabetes ethnomedicine. Methods: Bioassays of β-cell proliferation and insulin secretion as well as glucose diffusion as possible modes of action were recruited. Results: Similar to L-alanine insulinotropic efficacy in MIN6 β-cell, glucose-stimulated Ca2+ regulated- insulin secretion was potentiated by AEs of E.creticum (0.01 mg/ml and P.atlantica (0.01, 0.1 and 0.5 mg/ml. A.santolina AE, however, was found ineffective. Comparable to glucagon-like peptid-1-enhanced β-cell proliferation in 2-day treatment wells, a dose dependent augmentation of bromodeoxyuridine incorporation was obtained with the A.santolina AE (0.05-1 mg/ml, and E.creticum AE (0.1, 0.5 and 1 mg/ml. P.atlantica concentrations lacked pancreatic proliferative capacity. While A.santolina and E.creticum AEs proved inactive, P.atlantica inhibited dose dependently overnight glucose movement in vitro, as effectively as guar gum diffusional hindrance in a simple glucose dialysis model. Conclusion: Current findings signify the in vitro diverse therapeutic antidiabetes properties of the selected medicinal plants. Future directives may assess the use of A.santolina, E.creticum and P.atlantica as new potential sources of functional foods or nutraceuticals or active leads into diabetes type 2 pharmacotherapy. [J Exp Integr Med 2012; 2(3.000: 245-254

  1. Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice.

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    Antonino Sgroi

    Full Text Available BACKGROUND: Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration. METHODS: We performed 70%-hepatectomy in wild type (WT mice, IL-1ra knock-out (KO mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU incorporation, proliferating cell nuclear antigen (PCNA and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes. RESULTS: At 24h and at 48 h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1 and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment. CONCLUSION: IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.

  2. Chemotherapy-induced cognitive impairment is associated with decreases in cell proliferation and histone modifications

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    Briones Teresita L

    2011-12-01

    Full Text Available Abstract Background In this study, we examined the effects of cyclophosphamide, methothrexate, and 5-Fluorouracil (CMF drug combination on various aspects of learning and memory. We also examined the effects of CMF on cell proliferation and chromatin remodeling as possible underlying mechanisms to explain chemotherapy-associated cognitive dysfunction. Twenty-four adult female Wistar rats were included in the study and had minimitter implantation for continuous activity monitoring two weeks before the chemotherapy regimen was started. Once baseline activity data were collected, rats were randomly assigned to receive either CMF or saline injections given intraperitoneally. Treatments were given once a week for a total of 4 weeks. Two weeks after the last injection, rats were tested in the water maze for spatial learning and memory ability as well as discrimination learning. Bromodeoxyuridine (BrdU injection was given at 100 mg/Kg intraperitoneally 4 hours prior to euthanasia to determine hippocampal cell proliferation while histone acetylation and histone deacetylase activity was measured to determine CMF effects on chromatin remodeling. Results Our data showed learning and memory impairment following CMF administration independent of the drug effects on physical activity. In addition, CMF-treated rats showed decreased hippocampal cell proliferation, associated with increased histone acetylation and decreased histone deacetylase activity. Conclusions These results suggest the negative consequences of chemotherapy on brain function and that anti-cancer drugs can adversely affect the self-renewal potential of neural progenitor cells and also chromatin remodeling in the hippocampus. The significance of our findings lie on the possible usefulness of animal models in addressing the clinical phenomenon of 'chemobrain.'

  3. Detailed analysis of X chromosome inactivation in a 49,XXXXX pentasomy

    Science.gov (United States)

    Moraes, Lucia M; Cardoso, Leila CA; Moura, Vera LS; Moreira, Miguel AM; Menezes, Albert N; Llerena, Juan C; Seuánez, Héctor N

    2009-01-01

    Background Pentasomy X (49,XXXXX) has been associated with a severe clinical condition, presumably resulting from failure or disruption of X chromosome inactivation. Here we report that some human X chromosomes from a patient with 49,XXXXX pentasomy were functionally active following isolation in inter-specific (human-rodent) cell hybrids. A comparison with cytogenetic and molecular findings provided evidence that more than one active X chromosome was likely to be present in the cells of this patient, accounting for her abnormal phenotype. Results 5-bromodeoxyuridine (BrdU)-pulsed cultures showed different patterns among late replicating X chromosomes suggesting that their replication was asynchronic and likely to result in irregular inactivation. Genotyping of the proband and her mother identified four maternal and one paternal X chromosomes in the proband. It also identified the paternal X chromosome haplotype (P), indicating that origin of this X pentasomy resulted from two maternal, meiotic non-disjunctions. Analysis of the HUMANDREC region of the androgen receptor (AR) gene in the patient's mother showed a skewed inactivation pattern, while a similar analysis in the proband showed an active paternal X chromosome and preferentially inactivated X chromosomes carrying the 173 AR allele. Analyses of 33 cell hybrid cell lines selected in medium containing hypoxanthine, aminopterin and thymidine (HAT) allowed for the identification of three maternal X haplotypes (M1, M2 and MR) and showed that X chromosomes with the M1, M2 and P haplotypes were functionally active. In 27 cell hybrids in which more than one X haplotype were detected, analysis of X inactivation patterns provided evidence of preferential inactivation. Conclusion Our findings indicated that 12% of X chromosomes with the M1 haplotype, 43.5% of X chromosomes with the M2 haplotype, and 100% of the paternal X chromosome (with the P haplotype) were likely to be functionally active in the proband's cells, a

  4. Autologous Bone Marrow Mononuclear Cell Transplantation Delays Progression of Carotid Atherosclerosis in Rabbits.

    Science.gov (United States)

    Cui, Kefei; Ma, Xiao; Yu, Lie; Jiang, Chao; Fu, Chao; Fu, Xiaojie; Yu, Xiaofang; Huang, Yuanjing; Hou, Suyun; Si, Caifeng; Chen, Zhengguang; Yu, Jing; Wan, Jieru; Wang, Jian

    2016-09-01

    Bone marrow mononuclear cells (BMMNCs) can counteract oxidative stress and inhibit the inflammatory response in focal ischemic stroke models. However, the effect of BMMNC transplantation on carotid atherosclerosis needs to be determined. The carotid atherosclerotic plaque model was established in New Zealand White rabbits by balloon injury and 8 weeks of high-fat diet. Rabbits were randomized to receive an intravenous injection of autologous bromodeoxyuridine (BrdU)-labeled BMMNCs or an equal volume of phosphate-buffered saline. Plaques were evaluated for expression of proinflammatory and anti-inflammatory cytokines, anti-oxidant proteins, and markers of cell death. BMMNCs migrated into atherosclerotic plaque on the first day after cell transplantation. BMMNC-treated rabbits had smaller plaques and more collagen deposition than did the vehicle-treated controls on day 28 (p < 0.05). BMMNC treatment significantly increased endothelial nitric oxide synthase and the anti-oxidant enzymes glutathione peroxidase and superoxide dismutase in plaques compared to vehicle treatment on day 7. BMMNC-treated rabbits also had lower levels of cleaved caspase-3 expression; lower levels of proinflammatory cytokines interleukin-1β, tumor necrosis factor alpha, and matrix metalloproteinase 9; and higher levels of insulin-like growth factor-1 and its receptor (p < 0.05). Autologous BMMNC transplantation can suppress the process of atherosclerotic plaque formation and is associated with enhanced anti-oxidative effect, reduced levels of inflammatory cytokines and cleaved caspase-3, and increased expression of insulin-like growth factor-1 and its receptor. BMMNC transplantation represents a novel approach for the treatment of carotid atherosclerosis. PMID:26232064

  5. Effect of different BNCT protocols on DNA synthesis in precancerous and normal tissues in an experimental model of oral cancer

    International Nuclear Information System (INIS)

    We previously reported the therapeutic success of different BNCT protocols in the treatment of oral cancer, employing the hamster cheek pouch model. The aim of the present study was to evaluate the effect of these BNCT protocols on DNA synthesis in precancerous and normal tissue in this model and assess the potential lag in the development of second primary tumors in precancerous tissue. The data are relevant to potential control of field cancerized tissue and tolerance of normal tissue. We evaluated DNA synthesis in precancerous and normal pouch tissue 1-30 days post-BNCT mediated by BPA, GB-10 or BPA + GB-10 employing incorporation of bromo-deoxyuridine as an end-point. The BNCT-induced potential lag in the development of second primary tumors in precancerous tissue was monitored. A drastic, statistically significant reduction in DNA synthesis occurred in pacancerous tissue as early as 1 day post-BNCT and was sustained at virtually all time points until 30 days post-BNCT for all protocols. The histological categories evaluated individually within precancerous tissue (dysplasia, hyperplasia and NUMF [no unusual microscopic features]) responded similarly. DNA synthesis in normal tissue treated with BNCT oscillated around the very low pre-treatment values. A BNCT-induced lag in the development of second primary tumors was observed. BNCT induced a drastic fall in DNA synthesis in precancerous tissue that would be associated to the observed lag in the development of second primary tumors. The minimum variations in DNA synthesis in BNCT-treated normal tissue would correlate with the absence of normal tissue radiotoxicity. The present data would contribute to optimize therapeutic efficacy in the treatment of field-cancerized areas. (author)

  6. A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

    Directory of Open Access Journals (Sweden)

    Horváth Gábor V

    2010-01-01

    Full Text Available Abstract Background Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. Results Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. Conclusions In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

  7. A decrease in the addition of new cells in the nucleus accumbens and prefrontal cortex between puberty and adulthood in male rats.

    Science.gov (United States)

    Staffend, Nancy A; Mohr, Margaret A; DonCarlos, Lydia L; Sisk, Cheryl L

    2014-06-01

    Adolescence involves shifts in social behaviors, behavioral flexibility, and adaptive risk-taking that coincide with structural remodeling of the brain. We previously showed that new cells are added to brain regions associated with sexual behaviors, suggesting that cytogenesis may be a mechanism for acquiring adult-typical behaviors during adolescence. Whether pubertal cell addition occurs in brain regions associated with behavioral flexibility or motivation and whether these patterns differ between pubertal and adult animals had not been determined. Therefore, we assessed patterns of cell proliferation or survival in the prefrontal cortex and nucleus accumbens. Pubertal and adult male rats were given injections of bromo-deoxyuridine (BrdU). To assess cell proliferation, half of the animals from each group were sacrificed 24 h following the last injection. The remaining animals were sacrificed at Day 30 following the last injection to evaluate cell survival. Adult animals had significantly lower densities of BrdU-immunoreactive (ir) cells in the prefrontal cortex, irrespective of post-BrdU survival time, whereas in the nucleus accumbens, adult animals had a lower density of BrdU-ir cells at the short survival time; however, the density of BrdU-ir cells was equivalent in pubertal and adult animals at the longer survival time. These data provide evidence that cell addition during puberty may contribute to the remodeling of brain regions associated with behavioral flexibility and motivation, and this cell addition continues into adulthood, albeit at lower levels. Higher levels of cell proliferation or survival in younger animals may reflect a higher level of plasticity, possibly contributing to the dynamic remodeling of the pubertal brain. PMID:24339170

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

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    Cristina Rossi

    2014-09-01

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

  9. Agmatine increases proliferation of cultured hippocampal progenitor cells and hippocampal neurogenesis in chronically stressed mice

    Institute of Scientific and Technical Information of China (English)

    Yun-feng LI; Hong-xia CHEN; Ying LIU; You-zhi ZHANG; Yan-qin LIU; Jin LI

    2006-01-01

    Aim:To explore the mechanism of agmatine's antidepressant action.Methods: Male mice were subjected to a variety of unpredictable stressors on a daily basis over a 24-d period.The open-field behaviors of the mice were displayed and recorded using a Videomex-V image analytic system automatically.For bromodeoxyuridine (BrdU;thymidine analog as a marker for dividing cells) labeling,the mice were injected with BrdU (100 mg/kg,ip,twice per d for 2 d),and the hippocampal neurogenesis in stressed mice was measured by immunohistochemistry.The proliferation of cultured hippocampal progenitor cells from neonatal rats was determined by colorimetric assay (cell counting kit-8) and 3H-thymidine incorporation assay.Results:After the onset of chronic stress,the locomotor activity of the mice in the open field significantly decreased,while coadministration of agmatine 10 mg/kg (po) blocked it.Furthermore,the number of BrdU-labeled cells in the hippocampal dentate gyrus significantly decreased in chronically stressed mice, which was also blocked by chronic coadministration with agmatine 10 mg/kg (po). Four weeks after the BrdU injection, some of the new born cells matured and became neurons, as determined by double labeling for BrdU and neuron specific enolase (NSE), a marker for mature neurons.In vitro treatment with agmatine 0.1-10 μmo1/L for 3 d significantly increased the proliferation of the cultured hippocampal progenitor cells in a dose-dependent manner.Conclusion:We have found that agmatine increases proliferation of hippocampal progenitor cells in vitro and the hippocampal neurogenesis in vivo in chronically stressed mice.This may be one of the important mechanisms involved in agmatine's antidepressant action.

  10. Behavioral deficits induced by third-trimester equivalent alcohol exposure in male C57BL/6J mice are not associated with reduced adult hippocampal neurogenesis but are still rescued with voluntary exercise.

    Science.gov (United States)

    Hamilton, G F; Bucko, P J; Miller, D S; DeAngelis, R S; Krebs, C P; Rhodes, J S

    2016-11-01

    Prenatal alcohol exposure can produce permanent alterations in brain structure and profound behavioral deficits. Mouse models can help discover mechanisms and identify potentially useful interventions. This study examined long-term influences of either a single or repeated alcohol exposure during the third-trimester equivalent on survival of new neurons in the hippocampus, behavioral performance on the Passive avoidance and Rotarod tasks, and the potential role of exercise as a therapeutic intervention. C57BL/6J male mice received either saline or 5g/kg ethanol split into two s.c. injections, two hours apart, on postnatal day (PD)7 (Experiment 1) or on PD5, 7 and 9 (Experiment 2). All mice were weaned on PD21 and received either a running wheel or remained sedentary from PD35-PD80/81. From PD36-45, mice received i.p. injections of 50mg/kg bromodeoxyuridine (BrdU) to label dividing cells. Behavioral testing occurred between PD72-79. Number of surviving BrdU+ cells and immature neurons (doublecortin; DCX+) was measured at PD80-81. Alcohol did not affect number of BrdU+ or DCX+ cells in either experiment. Running significantly increased number of BrdU+ and DCX+ cells in both treatment groups. Alcohol-induced deficits on Rotarod performance and acquisition of the Passive avoidance task (Day 1) were evident only in Experiment 2 and running rescued these deficits. These data suggest neonatal alcohol exposure does not result in long-term impairments in adult hippocampal neurogenesis in the mouse model. Three doses of ethanol were necessary to induce behavioral deficits. Finally, the mechanisms by which exercise ameliorated the neonatal alcohol induced behavioral deficits remain unknown. PMID:27491590

  11. Developmental toxicity of dioxin to mouse embryonic teeth in vitro: arrest of tooth morphogenesis involves stimulation of apoptotic program in the dental epithelium

    International Nuclear Information System (INIS)

    Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can arrest molar tooth development in rats after in utero and lactational exposure, and that the sensitive stage is temporally restricted. To define the stage in which TCDD is able to arrest tooth development and the cellular background of the effect, mouse embryonic molar tooth explants including various early developmental stages from initiation to late cap stage were exposed to TCDD in organ culture. TCDD did not inhibit morphogenesis of the first molar teeth including the early bud-staged E12 first molars, but the teeth were smaller than in control cultures. Accordingly, the second molars underwent morphogenesis in the presence of TCDD when explanted at E15 when they were at the bud stage. TCDD arrested their development when explanted at E14 when they had not yet reached the early bud stage. Immunohistochemical localization of incorporated bromodeoxyuridine in cultured E14 teeth showed that TCDD did not affect cell proliferation. Localization of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL) method revealed that TCDD enhanced apoptosis of dental epithelial cells, especially in the dental lamina of both the first and second molars, and in the inner dental epithelium at the cusp tips of the first molars. Thus, TCDD can arrest tooth development in vitro if the exposure starts at the initiation stage, whereas exposure at later stages leads to smaller tooth size and deformation of cuspal morphology. TCDD interferes with tooth development by stimulating apoptosis in those cells of the dental epithelium, which are predetermined to undergo apoptosis during normal development

  12. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content

    Energy Technology Data Exchange (ETDEWEB)

    Pellicciari, C.; Mangiarotti, R.; Bottone, M.G.; Danova, M. [Univ. of Pavia (Italy); Wang, E. [Jewish General Hospital, Montreal, Quebec (Canada)

    1995-12-01

    Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G{sub 0}) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G{sub 0} cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G{sub 0} (statin positive) cells can be discriminated from the potentially cycling (statin negative) G{sub 1} cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G{sub 0}-G{sub 1} transition in unperturbed and drug-treated cell populations. 48 refs., 5 figs., 1 tab.

  13. MicroRNA mir-16 is anti-proliferative in enterocytes and exhibits diurnal rhythmicity in intestinal crypts

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishnan, Anita, E-mail: anita.balakrishnan@doctors.org.uk [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Department of Surgery, Harvard Medical School, Boston, MA 02115 (United States); School of Clinical Sciences, Division of Gastroenterology, University of Liverpool, Liverpool L69 3GE (United Kingdom); Stearns, Adam T. [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Department of Surgery, Harvard Medical School, Boston, MA 02115 (United States); Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 2JD (United Kingdom); Park, Peter J. [Department of Medicine, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Harvard Medical School, Center for Biomedical Informatics, Boston, MA 02115 (United States); Dreyfuss, Jonathan M. [Department of Medicine, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Ashley, Stanley W. [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Department of Surgery, Harvard Medical School, Boston, MA 02115 (United States); Rhoads, David B. [Department of Surgery, Harvard Medical School, Boston, MA 02115 (United States); Pediatric Endocrine Unit, MassGeneral Hospital for Children, Boston, MA 02114 (United States); Tavakkolizadeh, Ali, E-mail: atavakkoli@partners.org [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Department of Surgery, Harvard Medical School, Boston, MA 02115 (United States)

    2010-12-10

    Background and aims: The intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation. Methods: Diurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting. Results: mir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13-14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (cyclins D1-3, cyclin E1 and cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11 and 17 in antiphase to mir-16. Conclusions: This is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability.

  14. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    Energy Technology Data Exchange (ETDEWEB)

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-03-15

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  15. Trimethyltin intoxication induces the migration of ventricular/subventricular zone cells to the injured murine hippocampus.

    Science.gov (United States)

    Weig, Blair C; Richardson, Jason R; Lowndes, Herbert E; Reuhl, Kenneth R

    2016-05-01

    Following the postnatal decline of cell proliferation in the mammalian central nervous system, the adult brain retains progenitor cells with stem cell-like properties in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus. Brain injury can stimulate proliferation and redirect the migration pattern of SVZ precursor cells to the injury site. Sublethal exposure to the neurotoxicant trimethyltin (TMT) causes dose-dependent necrosis and apoptosis in the hippocampus dentate gyrus and increases SGZ stem cell proliferation to generate new granule cells. To determine whether SVZ cells also contribute to the repopulation of the TMT-damaged dentate gyrus, 6-8 week old male C3H mice were injected with the carbocyanine dye spDiI and bromodeoxyuridine (80mg/kg; ip.) to label ventricular cells prior to TMT exposure. The presence of labeled cells in hippocampus was determined 7 and 28days after TMT exposure. No significant change in the number of BrdU(+) and spDiI(+) cells was observed in the dentate gyrus 7days after TMT treatment. However, 28days after TMT treatment there was a 3-4 fold increase in the number of spDiI-labeled cells in the hippocampal hilus and dentate gyrus. Few spDiI(+) cells stained positive for the mature phenotypic markers NeuN or GFAP, suggesting they may represent undifferentiated cells. A small percentage of migrating cells were BrdU(+)/spDiI(+), indicating some newly produced, SVZ- derived precursors migrated to the hippocampus. Taken together, these data suggest that TMT-induced injury of the hippocampus can stimulate the migration of ventricular zone-derived cells to injured dentate gyrus. PMID:27045884

  16. Use of periodate-lysine-paraformaldehyde for the fixation of multiple antigens in human skin biopsies

    Directory of Open Access Journals (Sweden)

    L. Pieri

    2010-05-01

    Full Text Available Periodate-lysine-paraformaldehyde (PLP has been proposed as a fixative for glycoprotein antigens which should stabilize periodate oxidized polysaccharide chains through lysine mediated crosslinks, either directly or by the intermediation of formaldehyde. In spite of premises and attempts reported in the literature, this fixative has never become popular for the study of membrane antigens of immune system cells, which leads to doubts on its real efficacy. We have addressed this issue in biopsies of human skin and found that PLP followed by cryoprotection with 30% sucrose and cryosectioning, or PLP fixation of isolated epidermal sheets, consistently provided for good preservation of morphology and intense labeling of major histocompatibility complex class II molecules, CD1a, CD4, CD8, E-cadherin, cytokeratins in general, cytokeratin-18 in particular, and bromodeoxyuridine, incorporated by cycling cells in vitro, and for the demonstration of tyrosinase enzyme activity. PLP-fixed, osmicated and epon-embedded epidermal sheets proved as good as sheets fixed 365 with a mixture of formaldehyde and glutaraldehyde for electron microscopic morphological analysis. Also, these sheets were amenable to immunoperoxidase staining of Langerhans cell membrane antigen CD1a and keratinocyte membrane antigen E-cadherin before being osmicated and prepared for electron microscopy. In a parallel paper, we had also shown that oral mucosa biopsies fixed in PLP showed good morphology and immunolabeling of CD54, CD80, CD83 and CD86. Therefore, we conclude that PLP can be proposed as a multi-task fixative for light and electron microscopic analysis of membrane, cytoplasmic and nuclear antigens of immune system cells and keratinocytes.

  17. Spatial distribution of prominin-1 (CD133-positive cells within germinative zones of the vertebrate brain.

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    József Jászai

    Full Text Available BACKGROUND: In mammals, embryonic neural progenitors as well as adult neural stem cells can be prospectively isolated based on the cell surface expression of prominin-1 (CD133, a plasma membrane glycoprotein. In contrast, characterization of neural progenitors in non-mammalian vertebrates endowed with significant constitutive neurogenesis and inherent self-repair ability is hampered by the lack of suitable cell surface markers. Here, we have investigated whether prominin-1-orthologues of the major non-mammalian vertebrate model organisms show any degree of conservation as for their association with neurogenic geminative zones within the central nervous system (CNS as they do in mammals or associated with activated neural progenitors during provoked neurogenesis in the regenerating CNS. METHODS: We have recently identified prominin-1 orthologues from zebrafish, axolotl and chicken. The spatial distribution of prominin-1-positive cells--in comparison to those of mice--was mapped in the intact brain in these organisms by non-radioactive in situ hybridization combined with detection of proliferating neural progenitors, marked either by proliferating cell nuclear antigen or 5-bromo-deoxyuridine. Furthermore, distribution of prominin-1 transcripts was investigated in the regenerating spinal cord of injured axolotl. RESULTS: Remarkably, a conserved association of prominin-1 with germinative zones of the CNS was uncovered as manifested in a significant co-localization with cell proliferation markers during normal constitutive neurogenesis in all species investigated. Moreover, an enhanced expression of prominin-1 became evident associated with provoked, compensatory neurogenesis during the epimorphic regeneration of the axolotl spinal cord. Interestingly, significant prominin-1-expressing cell populations were also detected at distinct extraventricular (parenchymal locations in the CNS of all vertebrate species being suggestive of further, non

  18. Cytotoxic effects of curcumin in human retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Margrit Hollborn

    Full Text Available BACKGROUND: Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM and delayed apoptosis (above 1 µM. The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. CONCLUSION: It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as

  19. Prostate-Specific Natural Health Products (Dietary Supplements) Radiosensitize Normal Prostate Cells

    International Nuclear Information System (INIS)

    Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines. Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity was assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing γH2AX. Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2μg/mL], Trinovin [10μg/mL], and Prostate Rx [50 μg/mL]). However, both Trinovin (10μg/mL) and Prostate Rx (6μg/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair. Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells.

  20. Treatment with kaempferol suppresses breast cancer cell growth caused by estrogen and triclosan in cellular and xenograft breast cancer models.

    Science.gov (United States)

    Kim, Seung-Hee; Hwang, Kyung-A; Choi, Kyung-Chul

    2016-02-01

    As a phytoestrogen, kaempferol (Kaem) is one of bioflavonoids, which are found in a variety of vegetables including broccoli, tea and tomato. In this study, the antiproliferative effects of Kaem in triclosn (TCS)-induced cell growth were examined in MCF-7 breast cancer cells. TCS promoted the cell viability of MCF-7 cells via estrogen receptor α (ERα) as did 17β-estradiol (E2), a positive control. On the other hand, Kaem significantly suppressed E2 or TCS-induced cell growth. To elucidate the molecular mechanisms of TCS and Kaem, alterations in the expressions of cell cycle, apoptosis and metastasis-related genes were identified using western blot assay. The treatment of the cells with TCS up-regulated the protein expressions of cyclin D1, cyclin E and cathepsin D, while down-regulated p21 and bax expressions. Kaem reversed TCS-induced gene expressions in an opposite manner. The phosphorylation of IRS-1, AKT, MEK1/2 and ERK was increased by TCS, indicating that TCS induced MCF-7 cell proliferation via nongenomic ER signaling pathway associated with IGF-1R. Kaem presented an antagonistic activity on this signaling by down-regulating the protein expression of pIRS-1, pAkt and pMEK1/2 promoted by E2 or TCS. In an in vivo xenografted mouse model, tumor growth was induced by treatment with E2 or TCS, which was identified in the measurement of tumor volume, hematoxylin and eosin staining, bromodeoxyuridine and immunohistochemistry assay. On the contrary, E2 or TCS-induced breast tumor growth was inhibited by co-treatment with Kaem, which is consistent with in vitro results. Taken together, these results revealed that Kaem has an anticancer effect against procancer activity of E2 or TCS, a xenoestrogen, in breast cancer and may be suggested as a prominent agent to neutralize breast cancer risk caused by TCS. PMID:26878784

  1. Are newborn rat-derived neural stem cells more sensitive to lead neurotoxicity?

    Institute of Scientific and Technical Information of China (English)

    Yan Ho Chan; Mingyong Gao; Wutian Wu

    2013-01-01

    Lead ion (Pb2+) has been proven to be a neurotoxin due to its neurotoxicity on mammalian nervous system, especially for the developing brains of juveniles. However, many reported studies involved the negative effects of Pb2+ on adult neural cells of humans or other mammals, only few of which have examined the effects of Pb2+ on neural stem cells. The purpose of this study was to reveal the biological effects of Pb2+ from lead acetate [Pb (CH3COO)2] on viability, proliferation and differentiation of neural stem cells derived from the hippocampus of newborn rats aged 7 days and adult rats aged 90 days, respectively. This study was carried out in three parts. In the first part, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT viability assay) was used to detect the effects of Pb2+ on the cell viability of passage 2 hippocampal neural stem cells after 200 μM Pb2+, followed by immunocytochemical staining with anti-bromodeoxyuridine to demonstrate the effects of Pb2+ on cell proliferation. In the last part, passage 2 hippocampal neural Immunocytochemical staining with anti-microtubule-associated protein 2 (a neuron marker), anti-glial fibrillary acidic protein (an astrocyte marker), and anti-RIP (an oligodendrocyte marker) was performed to detect the differentiation commitment of affected neural stem cells after 6 days. The data showed that Pb2+ inhibited not only the viability and proliferation of rat hippocampal neural stem cells, but also their neuronal and oligodendrocyte differentiation in vitro. Moreover, increased activity of astrocyte differentiation of hippocampal neural stem cells from both newborn and adult rats was observed after exposure to high concentration of lead ion in vitro. These findings suggest that hippocampal neural stem cells of newborn rats were more sensitive than those from adult rats to Pb2+ cytotoxicity.

  2. Effects of co-administration of dietary sodium arsenate and 2,3-dimercaptopropane-1-sulfonic acid (DMPS) on the rat bladder epithelium

    International Nuclear Information System (INIS)

    Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. 2,3-Dimercaptopropane-1-sulfonic acid (DMPS), a chelating agent, is capable of reducing pentavalent arsenicals to the trivalent state and binding to the trivalent species, and it has been used in the treatment of heavy metal poisoning in humans. Therefore, we investigated the ability of DMPS to inhibit the cytotoxicity and regenerative urothelial cell proliferation induced by arsenate administration in vivo. Female rats were treated for 4 weeks with 100 ppm AsV. DMPS (2800 ppm) co-administered in the diet significantly reduced the AsV-induced cytotoxicity of superficial cells detected by scanning electron microscopy (SEM), and the incidence of simple hyperplasia observed by light microscopy and the bromodeoxyuridine (BrdU) labeling index. It also reduced the total concentration of arsenicals in the urine and the methylation of arsenic. There were no differences in oxidative stress as assessed by immunohistochemical staining for 8-hydroxy-2′-deoxyguanosine (8OHdG) of the bladder urothelium. No differences were detected in urine sediments between groups. These data suggest that DMPS has the ability to inhibit both arsenate-induced acute toxicity and regenerative proliferation of the rat bladder epithelium, most likely by decreasing exposure of the urothelium to trivalent arsenicals excreted in the urine. These data provide additional evidence that the effects of arsenate exposure in vivo do not appear to be related to oxidative effects on dG in DNA.

  3. Co-Mg-Al oxides issued of hydrotalcite precursors for total oxidation of volatile organic compounds. Identification and toxicological impact of the by-products

    Energy Technology Data Exchange (ETDEWEB)

    Gennequin, C.; Kouassi, S.; Tidahy, L.; Cousin, R.; Lamonier, J.F.; Garcon, G.; Shirali, P.; Cazier, F.; Aboukais, A.; Siffert, St. [Universite Lille Nord de France, 59 - Lille (France); Gennequin, C.; Kouassi, S.; Tidahy, L.; Cousin, R.; Lamonier, J.F.; Garcon, G.; Shirali, P.; Aboukais, A.; Siffert, St. [ULCO, UCEIV, MREI, 59 - Dunkerque (France); Cazier, F. [ULCO, CCM, MREI, 59 - Dunkerque (France)

    2010-05-15

    Catalysts based on Co-Mg-Al, which were used for the total oxidation of toluene, were synthesized by using the hydrotalcite pathway. The calcination allowed us to obtain various mixed oxide types (i.e. Co{sub 3}O{sub 4}, Co{sub 2}AlO{sub 4} or CoAl{sub 2}O{sub 4}), presenting meso-pores of about 8 nm and high specific surface areas. The solids were tested for the total oxidation of toluene and showed a total selectivity in CO{sub 2} and H{sub 2}O for 100% of toluene conversion. However, studies using diffuse reflectance infrared 'operando' and GC-MS allowed us to identify intermediary by-products stemming from the catalytic oxidation of toluene: benzene and small quantities of benzaldehyde, styrene and acetophenone. In order to contribute to the improvement of the current scientific knowledge on volatile organic compound (VOC) toxicity in humans, the lung toxicity of toluene, benzene or their association was determined by using a human epithelial lung cell model (i.e. L132 cell line). VOC cytotoxicity was studied with three complementary methods: the enzymatic activity of extracellular lactate dehydrogenase (LDH), the enzymatic activity of mitochondrial dehydrogenase (mDH), and the incorporation of 5-Bromodeoxyuridine (5-BrdU). Taken together, these results showed the occurrence of adverse effects, notably reported by significant increases in LDH activity in cell culture supernatants, 24 hours after L132 cell exposure not only to toluene alone or benzene alone, but also to their association. This original approach allowed us to integrate some toxicological parameters to help the choice of new-dedicated catalysts for the oxidative conversion of VOC. (authors)

  4. Utilization of the ex vivo LLNA: BrdU-ELISA to distinguish the sensitizers from irritants in respect of 3 end points-lymphocyte proliferation, ear swelling, and cytokine profiles.

    Science.gov (United States)

    Arancioglu, Seren; Ulker, Ozge Cemiloglu; Karakaya, Asuman

    2015-01-01

    Dermal exposure to chemicals may result in allergic or irritant contact dermatitis. In this study, we performed ex vivo local lymph node assay: bromodeoxyuridine-enzyme-linked immunosorbent assay (LLNA: BrdU-ELISA) to compare the differences between irritation and sensitization potency of some chemicals in terms of the 3 end points: lymphocyte proliferation, cytokine profiles (interleukin 2 [IL-2], interferon-γ (IFN-γ), IL-4, IL-5, IL-1, and tumor necrosis factor α [TNF-α]), and ear swelling. Different concentrations of the following well-known sensitizers and irritant chemicals were applied to mice: dinitrochlorobenzene, eugenol, isoeugenol, sodium lauryl sulfate (SLS), and croton oil. According to the lymph node results; the auricular lymph node weights and lymph node cell counts increased after application of both sensitizers and irritants in high concentrations. On the other hand, according to lymph node cell proliferation results, there was a 3-fold increase in proliferation of lymph node cells (stimulation index) for sensitizer chemicals and SLS in the applied concentrations; however, there was not a 3-fold increase for croton oil and negative control. The SLS gave a false-positive response. Cytokine analysis demonstrated that 4 cytokines including IL-2, IFN-γ, IL-4, and IL-5 were released in lymph node cell cultures, with a clear dose trend for sensitizers whereas only TNF-α was released in response to irritants. Taken together, our results suggest that the ex vivo LLNA: BrdU-ELISA method can be useful for discriminating irritants and allergens. PMID:25563296

  5. Possible participation of oxidative stress in causation of cell proliferation and in vivo mutagenicity in kidneys of gpt delta rats treated with potassium bromate

    International Nuclear Information System (INIS)

    Clarifying the participation of oxidative stress among possible contributing factors in potassium bromate (KBrO3)-induced carcinogenesis is of importance from the perspective of human health protection. In the present study, utilizing the antioxidative effects of α-tocopherol (α-TP) or sodium ascorbic acid (SAA) to attenuate oxidative stress, alterations in bromodeoxyuridine labeling indices (BrdU-LIs) and reporter gene mutations in kidneys of male and female gpt delta rats given KBrO3 were examined. Five male and female gpt delta rats in each group were given KBrO3 at a concentration of 500 ppm in the drinking water for 9 weeks, with 1% of α-TP or SAA administered in the diet from 1 week prior to the KBrO3 treatment until the end of the experiment. Increases in 8-hydroxydeoxyguanosine levels in kidney DNA of both sexes of rats given KBrO3 were significantly inhibited by SAA, but not α-TP. While BrdU-LIs in the proximal tubules of female rats were also significantly reduced by SAA, those in the males and gpt mutant frequencies in kidney DNA of both sexes were not affected by SAA or α-TP. Immunohistochemical and Western blot analyses for α2u-globulin strongly suggested that induction of cell proliferation observed in the males might primarily result from accumulation of this protein, independent of oxidative stress. The overall data indicated that while oxidative stress well correlates with induction of cell proliferation in females, its role in males and in generation of in vivo mutagenicity by KBrO3 in both sexes is limited

  6. Treatment with the herbal medicine, naoxintong improves the protective effect of high‑density lipoproteins on endothelial function in patients with type 2 diabetes.

    Science.gov (United States)

    Lv, Pu; Tong, Xunliang; Peng, Qing; Liu, Yuanyuan; Jin, Haiqiang; Liu, Ran; Sun, Wei; Pan, Bing; Zheng, Lemin; Huang, Yining

    2016-03-01

    The protective effect of high‑density lipoprotein (HDL) on endothelial function is impaired in patients with type 2 diabetes mellitus (T2DM), which may result in atherosclerotic complications. Naoxintong (NXT) is a compound preparation that includes Radix Astragali, Angelicae sinensis, Radix Paeoniae Rubra and Ligusticum wallichii. It is widely administered in China to prevent atherosclerotic complications. In the present study, NXT was administered to 69 patients with T2DM. HDLs were isolated from patient blood samples prior to and following the intervention. In vitro endothelial functions of HDL, including proliferation, migration, angiogenesis, and anti‑apoptosis were investigated by bromodeoxyuridine, wound healing, Transwell and Matrigel tube formation assays on human umbilical vein endothelial cells (HUVECs). The results from the present study demonstrated that HUVECs treated with HDL isolated from diabetic patients following NXT therapy exhibited increased proliferative effects (10‑27%; P<0.05), and improved migration ability (15‑35%; P<0.05), anti‑apoptotic function (23‑34%; P<0.05) and angiogenesis (30‑54%; P<0.001). Furthermore, the phosphorylation levels of Akt (26‑36%; P<0.01) and extracellular signal‑regulated kinase (16‑80%; P<0.01) were increased following NXT therapy. The present in vitro study demonstrates that the protective effect of HDL on endothelial function is markedly impaired in diabetic patients who tend to develop atherosclerosis, and the impaired function may be partly abrogated by NXT. PMID:26781332

  7. Sexual activity increases the number of newborn cells in the accessory olfactory bulb of male rats.

    Directory of Open Access Journals (Sweden)

    Wendy ePortillo

    2012-07-01

    Full Text Available In rodents, sexual behavior depends on the adequate detection of sexually relevant stimuli. The olfactory bulb (OB is a region of the adult mammalian brain undergoing constant cell renewal by continuous integration of new granular and periglomerular neurons in the accessory (AOB and main (MOB olfactory bulbs. The proliferation, migration, survival, maturation, and integration of these new cells to the OB depend on the stimulus that the subjects received. We have previously shown that 15 days after females control (paced the sexual interaction an increase in the number of cells is observed in the AOB. No changes are observed in the number of cells when females are not allowed to control the sexual interaction. In the present study we investigated if in male rats sexual behavior increases the number of new cells in the OB. Male rats were divided in five groups: 1 males that did not receive any sexual stimulation, 2 males that were exposed to female odors, 3 males that mated for 1 h and could not pace their sexual interaction, 4 males that paced their sexual interaction and ejaculated 1 time and 5 males that paced their sexual interaction and ejaculated 3 times. All males received three injections of the DNA synthesis marker bromodeoxyuridine at 1h intervals, starting 1h before the beginning of the behavioral test. Fifteen days later, males were sacrificed and the brains were processed to identify new cells and to evaluate if they differentiated into neurons. The number of newborn cells increased in the granular cell layer (also known as the internal cell layer of the AOB in males that ejaculated one or three times controlling (paced the rate of the sexual interaction. Some of these new cells were identified as neurons. In contrast, no significant differences were found in the mitral cell layer (also known as the external cell layer and glomerular cell layer of the AOB. In addition, no significant differences were found between groups in the MOB in

  8. The p150 subunit of CAF-1 causes association of SUMO2/3 with the DNA replication foci

    Energy Technology Data Exchange (ETDEWEB)

    Uwada, Junsuke [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Tanaka, Niina; Yamaguchi, Yutaro; Uchimura, Yasuhiro [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Shibahara, Kei-ichi [Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, Mishima (Japan); Nakao, Mitsuyoshi [Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan)

    2010-01-01

    The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to a wide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation. Nevertheless, it is unclear how SUMO2/3 and SUMO2/3-modified proteins are delivered to the chromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1), human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98-105 in p150 is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regions that replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA), and incorporation of bromodeoxyuridine (BrdU) were detected at foci co-localized with both p150 and SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficient in SUMO2/3 interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, our findings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3-modified proteins (or both) on chromatin fibers during replication.

  9. Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APP{sub swe}/PS1{sub {Delta}E9} transgenic mouse model of Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jun [Department of Medical Genetics, Third Military Medical University, Chongqing 400038 (China); Department of Physiology, Third Military Medical University, Chongqing 400038 (China); Song, Min; Wang, Yanyan [Department of Medical Genetics, Third Military Medical University, Chongqing 400038 (China); Fan, Xiaotang [Department of Histology and Embryology, Third Military Medical University, Chongqing 400038 (China); Xu, Haiwei, E-mail: haiweixu2001@yahoo.com.cn [Department of Physiology, Third Military Medical University, Chongqing 400038 (China); Bai, Yun, E-mail: baiyungene@gmail.com [Department of Medical Genetics, Third Military Medical University, Chongqing 400038 (China)

    2009-07-31

    In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP{sub swe}/PS1{sub {Delta}E9} mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP{sub swe}/PS1{sub {Delta}E9} transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.

  10. Allergy Enhances Neurogenesis and Modulates Microglial Activation in the Hippocampus

    Science.gov (United States)

    Klein, Barbara; Mrowetz, Heike; Thalhamer, Josef; Scheiblhofer, Sandra; Weiss, Richard; Aigner, Ludwig

    2016-01-01

    Allergies and their characteristic TH2-polarized inflammatory reactions affect a substantial part of the population. Since there is increasing evidence that the immune system modulates plasticity and function of the central nervous system (CNS), we investigated the effects of allergic lung inflammation on the hippocampus—a region of cellular plasticity in the adult brain. The focus of the present study was on microglia, the resident immune cells of the CNS, and on hippocampal neurogenesis, i.e., the generation of new neurons. C57BL/6 mice were sensitized with a clinically relevant allergen derived from timothy grass pollen (Phl p 5). As expected, allergic sensitization induced high serum levels of allergen-specific immunoglobulins (IgG1 and IgE) and of TH2 cytokines (IL-5 and IL-13). Surprisingly, fewer Iba1+ microglia were found in the granular layer (GL) and subgranular zone (SGZ) of the hippocampal dentate gyrus and also the number of Iba1+MHCII+ cells was lower, indicating a reduced microglial surveillance and activation in the hippocampus of allergic mice. Neurogenesis was analyzed by labeling of proliferating cells with bromodeoxyuridine (BrdU) and determining their fate 4 weeks later, and by quantitative analysis of young immature neurons, i.e., cells expressing doublecortin (DCX). The number of DCX+ cells was clearly increased in the allergy animals. Moreover, there were more BrdU+ cells present in the hippocampus of allergic mice, and these newly born cells had differentiated into neurons as indicated by a higher number of BrdU+NeuN+ cells. In summary, allergy led to a reduced microglia presence and activity and to an elevated level of neurogenesis in the hippocampus. This effect was apparently specific to the hippocampus, as we did not observe these alterations in the subventricular zone (SVZ)/olfactory bulb (OB) system, also a region of high cellular plasticity and adult neurogenesis.

  11. Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation.

    Directory of Open Access Journals (Sweden)

    XiaoTong Yu

    Full Text Available Urotensin II (UII, a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC, and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE or 2', 7'-dichlorofluorescein diacetate (DCF-DA fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC.

  12. Interactions with the young down-regulate adult olfactory neurogenesis and enhance the maturation of olfactory neuroblasts in sheep mothers.

    Science.gov (United States)

    Brus, Maïna; Meurisse, Maryse; Keller, Matthieu; Lévy, Frédéric

    2014-01-01

    New neurons are continuously added in the dentate gyrus (DG) and the olfactory bulb of mammalian brain. While numerous environmental factors controlling survival of newborn neurons have been extensively studied, regulation by social interactions is less documented. We addressed this question by investigating the influence of parturition and interactions with the young on neurogenesis in sheep mothers. Using Bromodeoxyuridine, a marker of cell division, in combination with markers of neuronal maturation, the percentage of neuroblasts and new mature neurons in the olfactory bulb and the DG was compared between groups of parturient ewes which could interact or not with their lamb, and virgins. In addition, a morphological analysis was performed by measuring the dendritic arbor of neuroblasts in both structures. We showed that the postpartum period was associated with a decrease in olfactory and hippocampal adult neurogenesis. In the olfactory bulb, the suppressive effect on neuroblasts was dependent on interactions with the young whereas in the DG the decrease in new mature neurons was associated with parturition. In addition, dendritic length and number of nodes of neuroblasts were significantly enhanced by interactions with the lamb in the olfactory bulb but not in the DG. Because interactions with the young involved learning of the olfactory signature of the lamb, we hypothesize that this learning is associated with a down-regulation in olfactory neurogenesis and an enhancement of olfactory neuroblast maturation. Our assumption is that fewer new neurons decrease cell competition in the olfactory bulb and enhance maturation of those new neurons selected to participate in the learning of the young odor. PMID:24600367

  13. Controls over fungal communities and consequences for nutrient cycling

    Science.gov (United States)

    Treseder, K. K.; Majumder, P.; Bent, E.; Borneman, J.; Allison, S. D.; Hanson, C. A.

    2007-12-01

    Soils harbor a high diversity of microbes-- as many as 100 species of fungi within a square meter. If different species target different components of litter, a more diverse community of fungi should lead to faster decomposition rates. We examined the hypotheses that variation in substrate use among fungal groups and variation in nitrogen availability are both important controls over the diversity of fungi in an Alaskan boreal forest. Nitrogen availability was considered because microbes are often N-limited, and because humans are altering N availability via anthropogenic N deposition and global warming. We used nucleotide analogs to link fungal groups with their role in decomposition in field samples. Leaf litter collected from the forest floor was supplemented with one of four N-containing compounds. Bromodeoxyuridine (BrdU, a thymidine analog) was also added. After 48 hours incubation, DNA was extracted. Most growing fungi should have assimilated the BrdU into new DNA. Their genetic identity was determined using oligonucleotide fingerprinting of rRNA genes (OFRG). OFRG is an rRNA gene profiling method that sorts genes into taxonomic groups with a high degree of resolution, and has a large capacity for sample processing. Fungal groups that proliferated following the addition of a given compound probably metabolized that compound. We found that fungal taxa varied in their responses to different substrates, indicating that they differed in substrate use. Specifically, community composition of fungi was significantly different among substrate treatments (P tannin-protein complexes (P = 0.014); Saccharomycetales, arginine (P = 0.042); and Polyporales, arginine and lignocellulose (P = 0.040). In a complementary experiment, we used BrdU labeling to characterize effects of N fertilization on fungal community composition. We observed that N fertilization decreased the richness of fungal taxa by 22%. Helotiales and Saccharomycetales tended to increase under N

  14. Epigallocathechin gallate, polyphenol present in green tea, inhibits stem-like characteristics and epithelial-mesenchymal transition in nasopharyngeal cancer cell lines

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    Lin Chien-Hung

    2012-10-01

    Full Text Available Abstract Background Previous studies have demonstrated that the consumption of green tea inhibits the growth of various cancers. Most cancers are believed to be initiated from and maintained by a small population of cancer stem-like cells (CSC or tumor-initiating cells (TIC that are responsible for tumor relapse and chemotherapeutic resistance. Although epigallocathechin gallate (EGCG, the most abundant catechin in green tea, has been reported to induce growth inhibition and apoptosis in some cancer cells, its effect on CSC is undefined. In this study, we enriched CSC by the sphere formation, and provided an efficient model for further experiments. Using this method, we examined the effects of EGCG regulating the nasopharyngeal carcinoma (NPC CSC and attempted to elucidate the possible mechanisms. Methods NPC TW01 and TW06 cell lines were enriched by sphere formation and characterized their phenotypical properties, such as invasion capacity, epithelial-mesenchymal transition (EMT and gene expression were analyzed by quantitative real-time reverse transcription polymerase chain reaction (q-RT-PCR. EGCG-induced growth inhibition in the parental and sphere-derived cells was determined by MTT and bromodeoxyuridine (BrdU assay. EGCG-induced apoptosis was analyzed by flow cytometry with Annexin V and PI staining. The effects of EGCG on sphere-derived cell tumorigenicity, migration and invasion were determined by soft agar assay, wound healing, and cell invasion assay. The alternation of protein expression regulated by EGCG on these sphere-derived cells was assessed by immunofluorescence staining and western blot. Results NPC sphere-derived cells grown in serum-free non-adherent culture showed increased expression of stem cell markers and EMT markers compared to parental cells grown in conventional culture. Although EGCG induced growth inhibition and apoptosis in the parental cells in a dose-dependent manner, it was not as effective against spheres

  15. MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.

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    Dan Yu

    Full Text Available Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC proliferation with little effect on endothelial cell (EC proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS, cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated

  16. Effect of dietary treatment with dimethylarsinous acid (DMAIII) on the urinary bladder epithelium of arsenic (+3 oxidation state) methyltransferase (As3mt) knockout and C57BL/6 wild type female mice

    International Nuclear Information System (INIS)

    Highlights: ► As3mt KO and WT mice were treated with DMAIII for 4 weeks in drinking water. ► No treatment-related death or whole body toxicity observed in any of the groups. ► Urothelium showed simple hyperplasia in treated KO and WT mice. ► BrdU labeling index of urothelium was significantly increased in treated KO mice. - Abstract: Chronic exposure to inorganic arsenic (iAs) is carcinogenic to the human urinary bladder. It produces urothelial cytotoxicity and proliferation in rats and mice. DMAV, a major methylated urinary metabolite of iAs, is a rat bladder carcinogen, but without effects on the mouse urothelium. DMAIII was shown to be the likely urinary metabolite of DMAV inducing urothelial changes and is also postulated to be one of the active metabolites of iAs. To evaluate potential DMAIII-induced urothelial effects, it was administered to As3mt knockout mice which cannot methylate arsenicals. Female C57BL/6 wild type and As3mt knockout mice (10/group) were administered DMAIII, 77.3 ppm in water for four weeks. Urothelial effects were evaluated by light and scanning electron microscopy (EM) and immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation. EM findings were rated 1–5, with higher rating indicating greater extent of cytotoxicity visualized. DMAIII significantly increased the BrdU labeling index, a ratio of BrdU labeled cells to non-labeled cells, in the treated knockout group compared to control and wild type treated groups. DMAIII induced simple hyperplasia in more knockout mice (4/10) compared to wild type mice (2/10). All treated knockout mice had more and larger intracytoplasmic granules compared to the treated wild type mice. Changes in EM classification were not significant. In conclusion, DMAIII induces urothelial toxicity and regenerative hyperplasia in mice and most likely plays a role in inorganic arsenic-induced urothelial changes. However, DMAV does not induce hyperplasia in mice, suggesting that urinary

  17. GIT1 is a novel prognostic biomarker and facilitates tumor progression via activating ERK/MMP9 signaling in hepatocellular carcinoma

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    Chen J

    2015-12-01

    Full Text Available Junyi Chen,1,* Pinghua Yang,2,* Jue Yang,2 Zhijian Wen,2 Baohua Zhang,2 Xin Zheng1,3 1Department of General Surgery, Branch of the First People’s Hospital of Shanghai, 2Department of Hepatic Surgery, the Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 3Department of Traditional Chinese Medicine, Branch of the first People’s Hospital of Shanghai, Shanghai, People’s Republic of China *These authors contributed equally to this work Aim: Multiple studies have revealed that G-protein-coupled receptor kinase-interacting protein 1 (GIT1 is overexpressed in many cancers and facilitates tumor progression. However, the role of GIT1 in hepatocellular carcinoma (HCC remains unclear.Methods: GIT1 expression was detected in cell lines and 130 pairs of HCC and matched adjacent noncancerous samples. Transwell assay, flow cytometry, caspase 3/7 ­activity assay, 5-bromodeoxyuridine cell proliferation assay, and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay were used to assess invasion, migration, apoptosis, and proliferation of HCC cells. Furthermore, GIT1 expression was detected by immunohistochemistry to evaluate its correlation with phospho-extracellular signal-regulated kinase (p-ERK1/2. The regulatory effect of GIT1 on ERK1/2, p-ERK1/2, and matrix metalloproteinase-9 (MMP9 in HCC cells was confirmed by immunoblotting.Results: In this study, we demonstrated that GIT1 was more highly expressed in HCC samples than that in non-HCC samples, and overexpression of GIT1 was correlated with clinicopathological features of poor prognosis. Clinical analysis demonstrated that GIT1 is an independent prognostic biomarker for predicting overall survival and disease-free survival of patients with HCC. In vitro studies showed that downregulation of GIT1 facilitated HCC cell apoptosis and repressed HCC cell invasion, migration, and proliferation. Overexpression of GIT1 is associated with p-ERK1/2 amplification in HCC

  18. Gliadin-mediated proliferation and innate immune activation in celiac disease are due to alterations in vesicular trafficking.

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    M Vittoria Barone

    Full Text Available BACKGROUND AND OBJECTIVES: Damage to intestinal mucosa in celiac disease (CD is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. METHODS/PRINCIPAL FINDINGS: Cell proliferation was evaluated by bromodeoxyuridine (BrdU incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R alpha, did not require new protein synthesis and functioned as a growth factor. CONCLUSION: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

  19. Migration and differentiation of bone marrow-derived multipotent adult progenitor cells through tail vein injection in a rat model of cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Lei Lei; Ruixiang Zhou

    2009-01-01

    BACKGROUND: Multipotent adult progenitor cells (MAPCs) from the bone marrow have been shown to differentiate into neurons.OBJECTIVE: To observe migration, survival, and neuronal-like differentiation of MAPCs by tail vein injection.DESIGN, TIME AND SETTING: Randomized, controlled experiment of neural tissue engineering was performed at the Laboratory for Cardio-Cerebrovascular Disease, Hospital of Integrated Traditional and Western Medicine, Tongji Medical College of Huazhong University of Science and Technology between September 2006 and August 2007.MATERIALS: Eighty Sprague Dawley rats, 3-6 months old, underwent cerebral ischemia/reperfusion by thread technique, and were randomly divided into model and MAPCs groups (n = 40).METHODS: Mononuclear cells were harvested from bone marrow using the Ficoll-Paque density gradient centrifugation method. After removing CD45 and glycophorin A-positive cells (GLYA+) with immunomagnetic beads, CD45 GLYA adult progenitor cells were labeled with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). A total of 1 mL cell suspension, containing 5 ×106 MAPCs, was injected into the MAPCs group through the tail vein. A total of 1 mL normal saline was injected into the model rats.MAIN OUTCOME MEASURES: After 60 days, BrdU and neuron-specific enolase double-positive cells were observed using immunofluorescence. Cell morphology was observed under electron microscopy, and nerve growth factor mRNA was measured through RT-PCR. In addition, rat neurological functions were measured with behavioral tests.RESULTS: Immunofluorescence revealed that MAPCs positive for BrdU and neuron specific enolase were found surrounding the ischemic focus in the MAPCs group. Microscopic observation suggested that MAPCs-derived neuronal-like cells connected with other nerve cells to form synapses, Compared with the model animals, the level of nerve growth factor mRNA was significantly upregulated in rats injected with MAPCs (P < 0.05). In addition, rats in the MAPCs

  20. Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

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    Fiona L Cousins

    Full Text Available BACKGROUND: In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. METHODOLOGY: A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4 withdrawal; mice received a single injection of bromodeoxyuridine (BrdU 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. PRINCIPAL FINDINGS: Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. CONCLUSIONS/SIGNIFICANCE: These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and

  1. Strategies for repair of white matter: Influence of osmolarity and microglia on proliferation and apoptosis of oligodendrocyte precursor cells in different basal culture media

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    Karolina eKleinsimlinghaus

    2013-12-01

    Full Text Available The aim of the present study has been to obtain high yields of oligodendrocyte precursor cells (OPCs in culture. This is a first step in facilitation of myelin repair. We show that, in addition to factors, known to promote proliferation, such as basic fibroblast growth factor (FGF-2 and platelet derived growth factor (PDGF the choice of the basal medium exerts a significant influence on the yield of OPCs in cultures from newborn rats. During a culture period of up to 9 days we observed larger numbers of surviving cells in Dulbecco’s Modified Eagle Medium (DMEM and Roswell Park Memorial Institute Medium (RPMI compared with Neurobasal Medium (NB. A larger number of A2B5-positive OPCs was found after 6 days in RPMI based media compared with NB. The percentage of bromodeoxyuridine (BrdU-positive cells was largest in cultures maintained in DMEM and RPMI. The percentage of caspase-3 positive cells was largest in NB, suggesting that this medium inhibits OPC proliferation and favors apoptosis. A difference between NB and DMEM as well as RPMI is the reduced Na+-content. The addition of equiosmolar supplements of mannitol or NaCl to NB medium rescued the BrdU-incorporation rate. This suggested that the osmolarity influences the proliferation of OPCs. Plating density as well as residual microglia influence OPC survival, BrdU incorporation and caspase-3 expression. We found, that high density cultures secrete factors that inhibit BrdU incorporation whereas the presence of additional microglia induces an increase in caspase-3 positive cells, indicative of enhanced apoptosis. An enhanced number of microglia could thus also explain the stronger inhibition of OPC differentiation observed in high density cultures in response to treatment with the cytokines TNF-a and IFN-g.We conclude that a maximal yield of OPCs is obtained in a medium of an osmolarity higher than 280 mOsm plated at a relatively low density in the presence of as little microglia as technically

  2. Characterization of the early proliferative response of the rodent bladder to subtotal cystectomy: a unique model of mammalian organ regeneration.

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    Charles C Peyton

    Full Text Available Subtotal cystectomy (STC; surgical removal of ∼75% of the rat urinary bladder elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU. Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP, muscularis propria (MP and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA, to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh, Gli-1 and bone morphogenic factor-4 (BMP-4 were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends

  3. 缬草对慢性应激导致的抑郁大鼠大脑海马5-羟色胺水平、细胞增殖及神经元数量的影响%Effects of Valerian on the level of 5-hydroxytryptamine, cell proliferation and neurons in cerebral hippocampus of rats with depression induced by chronic mild stress

    Institute of Scientific and Technical Information of China (English)

    唐久余; 曾园山; 陈巧格; 秦雅静; 陈穗君; 钟志强

    2008-01-01

    目的:探讨缬草对慢性应激导致的抑郁大鼠大脑海马5-羟色胺(5-hydroxytryptamine,5-HT)水平、细胞增殖及神经元数量的影响.方法:70只大鼠被分成正常对照组、未用药模型组、阴性对照模型组、阳性对照模型组和低、中、高剂量缬草治疗组,每组10只.除正常对照组外,给予其余6组大鼠4周慢性应激建立抑郁症模型.除未用药模型组大鼠正常饲养外,其余6组大鼠在模型建立后分别灌服5%羧甲基纤维素钠、氟西汀以及低、中、高剂量缬草,灌药周期均为3周.灌药结束后,体内注射5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)标记海马增殖细胞,应用高效液相色谱法检测海马组织5-羟色胺水平,采用形态计量学方法计数海马神经元数量.结果:与正常对照组比较,低、中剂量缬草治疗组的海马5-HT含量增加,并恢复至正常水平.灌服低剂量缬草3周后,抑郁大鼠海马BrdU阳性细胞数目和神经元数量恢复性增加至正常对照组大鼠的水平.结论:小剂量缬草能够促进抑郁大鼠大脑海马5-HT水平及细胞增殖数量恢复至正常状况,同时具有保护海马受损伤神经元的作用.

  4. Use of 5-[76Br]bromo-2'-fluoro-2'-deoxyuridine as a ligand for tumour proliferation: validation in an animal tumour model

    International Nuclear Information System (INIS)

    Uncontrolled cell proliferation is one of the prominent features in cancer development. Precise tools are needed for determination of the proliferation rate before, during and after treatment, thereby permitting assessment of treatment efficacy. The purpose of this study was to validate the use of 5-[76Br]bromo-2'-fluoro-2'-deoxyuridine (76Br-BFU) as a proliferation marker in an animal tumour model. Comparison was made with 2-[14C]thymidine (14C-TdR) incorporation and the labelling index assessed by bromodeoxyuridine (BrdUrd-LI). Fibrosarcoma (NFSA)-bearing mice were used for all experiments. Gemcitabine (dFdC), a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. dFdC was injected intraperitoneally at a dose of 0.5 mg/kg or 40 mg/kg to induce partial (∼50%) or complete inhibition of DNA synthesis, respectively. 76Br-BFU (0.5-3 MBq per animal), 14C-TdR (37-74 kBq per animal) and cold BrdUrd (60 mg/kg) were injected intraperitoneally in combination or alone. Animals were sacrificed at various times after tracer administration, and tumour and small intestine were removed for determination of radioactivity in whole tissue and the DNA fraction, as well as for LI assessment by flow cytometry. Cimetidine (6 mg/kg) was used to decrease 76Br-BFU elimination and increase its bioavailability. The fraction of radioactivity associated with DNA increased with the time interval between tracer injection and tissue removal. At 6 h after injection, for both tracers, more than 95% of the radioactivity in the tumours was associated with the DNA fraction and an excellent correlation was observed with the LI. Similar findings were observed in the small intestine. Under all experimental conditions, 76Br-BFU uptake was 4-10 times lower than 14C-TdR uptake. Co-injection of cimetidine resulted in a three- to fourfold increase in 76Br-BFU incorporation without affecting the effect of dFdC on DNA synthesis. 76Br-BFU is a potentially good tracer for the assessment

  5. Impaired glutathione redox system paradoxically suppresses angiotensin II-induced vascular remodeling.

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    Kazuma Izawa

    Full Text Available BACKGROUND: Angiotensin II (AII plays a central role in vascular remodeling via oxidative stress. However, the interaction between AII and reduced glutathione (GSH redox status in cardiovascular remodeling remains unknown. METHODS: In vivo: The cuff-induced vascular injury model was applied to Sprague Dawley rats. Then we administered saline or a GSH inhibitor, buthionine sulfoximine (BSO, 30 mmol/L in drinking water for a week, subsequently administered 4 more weeks by osmotic pump with saline or AII (200 ng/kg/minute to the rats. In vitro: Incorporation of bromodeoxyuridine (BrdU was measured to determine DNA synthesis in cultured rat vascular smooth muscle cells (VSMCs. RESULTS: BSO reduced whole blood GSH levels. Systolic blood pressure was increased up to 215 ± 4 mmHg by AII at 4 weeks (p<0.01, which was not affected by BSO. Superoxide production in vascular wall was increased by AII and BSO alone, and was markedly enhanced by AII+BSO. The left ventricular weight to body weight ratio was significantly increased in AII and AII+BSO as compared to controls (2.52 ± 0.08, 2.50 ± 0.09 and 2.10 ± 0.07 mg/g respectively, p<0.05. Surprisingly, the co-treatment of BSO totally abolished these morphological changes. Although the vascular circumferential wall stress was well compensated in AII, significantly increased in AII+BSO. The anti-single-stranded DNA staining revealed increasing apoptotic cells in the neointima of injured arteries in BSO groups. BrdU incorporation in cultured VSMCs with AII was increased dose-dependently. Furthermore it was totally abolished by BSO and was reversed by GSH monoethyl ester. CONCLUSIONS: We demonstrated that a vast oxidative stress in impaired GSH redox system totally abolished AII-induced vascular, not cardiac remodeling via enhancement of apoptosis in the neointima and suppression of cell growth in the media. The drastic suppression of remodeling may result in fragile vasculature intolerable to mechanical

  6. LXA{sub 4} actions direct fibroblast function and wound closure

    Energy Technology Data Exchange (ETDEWEB)

    Herrera, Bruno S. [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Leung, Kai P., E-mail: kai.p.leung.civ@mail.mil [Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Van Dyke, Thomas E., E-mail: tvandyke@forsyth.org [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States)

    2015-09-04

    Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A{sub 4} (LXA{sub 4}), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA{sub 4} on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA{sub 4} receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA{sub 4} receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA{sub 4} slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA{sub 4} tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA{sub 4} in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF

  7. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations.

    Science.gov (United States)

    Natarajan, A T; van Zeeland, A A; Zwanenburg, T S

    1983-01-01

    The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, we have measured the influence of 3AB on DNA repair following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. Both acentric fragments and exchanges increase indicating that the presence of 3AB slows down the repair of DNA strand breaks (probably DNA double strand breaks), thus making breaks available for interaction with each other to give rise to exchanges. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. Most 3AB induced SCEs occur during the second cell cycle, in which DNA containing bromouridine (BU) is used as template for replication. BU containing DNA appears to be prone to errors during replication. The extent of increase in the frequencies of SCEs by 3AB is correlated with the amount of BU incorporated in the DNA of the cells. The frequencies of spontaneously occurring DNA single strand breaks in cells grown in BrdUrd containing medium are higher than in the cells grown in normal medium and this increase depends on the amount of BU incorporated in the DNA of these cells. We have studied the extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the

  8. Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single kidney in the pig

    International Nuclear Information System (INIS)

    Purpose: Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single pig kidney were assessed. Methods and Materials: The right kidney of 13 adult female Large White pigs was irradiated with a single dose of 9.8 Gy γ rays. Animals were serially killed between 2 and 24 weeks postirradiation (PI); 1 h prior to postmortem each pig received 500 mg bromodeoxyuridine (BrdUrd). At postmortem, both kidneys were removed and tissue taken to prepare cell suspensions. The labeling index (LI) of these suspensions was measured using flow cytometry; in vivo BrdUrd incorporation in glomerular and tubular cells was determined immunohistochemically. The kidneys were also assessed histologically. Results: Irradiation of the right kidney alone resulted in a significant increase in renal cell LI in both the irradiated and the contralateral unirradiated kidney within 2 weeks of irradiation; peak values of 1.57 ± 0.32% and 1.04 ± 0.13%, respectively, were seen 4 weeks PI, significantly greater p < 0.001) than the preirradiation value of 0.18 ± 0.01%. The LI values then declined with time, but remained greater than those seen prior to irradiation. A similar pattern of response was determined from counts of labeled glomerular and tubular cells identified immunohistochemically. The increase in labeled glomerular cells was seen 2 weeks PI, whereas that for the tubular cells did not occur until 4 weeks PI. The irradiated kidney exhibited diffuse, progressive glomerular alterations. In contrast, tubular damage was focal; the irradiated kidney also exhibited a prominent vasculopathy, involving arteriolar and peripheral interlobular artery thickening. The contralateral unirradiated kidney appeared unchanged. Conclusion: These findings confirm the hypothesis that the morphologic and kinetic responses observed after irradiation of a single kidney are similar to those observed after irradiation of both kidneys. Renal irradiation results in

  9. Resveratrol, but not EGCG, in the diet suppresses DMBA-induced mammary cancer in rats

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    Whitsett Timothy

    2006-05-01

    Full Text Available Abstract Despite the advent of new and aggressive therapeutics, breast cancer remains a leading killer among women; hence there is a need for the prevention of this disease. Several naturally occurring polyphenols have received much attention for their health benefits, including anti-carcinogenic properties. Two of these are resveratrol, a component of red grapes, and epigallocatechin-3-gallate (EGCG, the major catechin found in green tea. In this study, we tested the hypothesis that these two polyphenols protect against chemically-induced mammary cancer by modulating mammary gland architecture, cell proliferation, and apoptosis. Female Sprague-Dawley CD rats were exposed to either resveratrol (1 g/kg AIN-76A diet, EGCG (0.065% in the drinking water, or control diet (AIN-76A for the entirety of their life starting at birth. At 50 days postpartum, rats were treated with 60 mg dimethylbenz[a]anthracene (DMBA/kg body weight to induce mammary cancer. Resveratrol, but not EGCG, suppressed mammary carcinogenesis (fewer tumors per rat and longer tumor latency. Analysis of mammary whole mounts from 50-day-old rats revealed that resveratrol, but not EGCG, treatment resulted in more differentiated lobular structures. Bromodeoxyuridine (BrdU incorporation studies showed that resveratrol treatment caused a significant reduction in proliferative cells in mammary terminal ductal structures at 50 days postpartum, making them less susceptible to carcinogen insult. The epithelial cells of terminal end buds in the mammary glands of resveratrol-treated rats also showed an increase in apoptotic cells compared to the control or EGCG-treated rats as measured by a DNA fragmentation assay. At the given doses, resveratrol treatment resulted in a serum resveratrol concentration of 2.00 μM, while treatment with EGCG resulted in a serum EGCG concentration of 31.06 nM. 17β-Estradiol, progesterone, and prolactin concentrations in the serum were not significantly affected

  10. [Novel calretinin-positive cells with polymorphous spines in the mouse forebrain during early postnatal ontogenesis].

    Science.gov (United States)

    Revishchin, A V; Okhotin, V E; Pavlova, G V

    2009-01-01

    Using an immunocytochemical method for calretinin (CR) detection, we have earlier described (Morfologiya, 2009 v. 135. No. 3, p. 7-19) the population of previously unknown mono- and bipolar cells with polymorphous spines (PS) covering their cell bodies and processes, in adult mice forebrain structures adjacent to anterior horn of lateral ventricle. CR-positive spiny (CR+PS) cells were negative to GAD67 and were detected in the white matter and in layers V and VI of frontal area of dorsomedial cortex close to the cingulum, in in rostro-dorsal part of the caudate nucleus-putamen complex, anterior olfactory nucleus and in subependymal layer of the dorso-lateral angle of the lateral ventricle. In this work, the distribution of these cells in 7-day-old mice was studied. Comparative topographical analysis of definitive and early CR+PS cells demonstrated that in 7-day-old mice CR+PS cells were absent from the areas of their localization in adult animals - anterior olfactory nucleus, cortical plate and inner portion of neostriatum. Meanwhile, some CR+PS-like cells were detected in 7-day-old mice inside the rostral migratory route, close to neostriatum anterior boundary, along the dorsal border between neostriatum and corpus callosum, subependymal layer of lateral wall of the lateral ventricle, and in the cingulum area. These findings are indicative of the possible postnatal appearance of CR+PS cells. To test this hypothesis, the experiments were conducted in which bromodeoxyuridine (BrdU) was administered to the mice on their postnatal days 2-4 with the subsequent study of the brain sections of these animals sacrificed on their postnatal day 20. Double immunolabeling of these sections for CR and BrdU has detected the presence of CR+PS cells that contained postnatally administered BrdU. These results strongly suggest that, at least, some portion of CR+PS cells have their mitosis postnatally. It may be assumed, that CR+PS cells migrate to the sites of their distribution in

  11. New calretinin-positive cells with polymorphous spines in the mouse forebrain during early postnatal ontogeny.

    Science.gov (United States)

    Revishchin, A V; Okhotin, V E; Pavlova, G V

    2010-10-01

    Immunohistochemical studies of calretinin (CR) in forebrain structures adjacent to the anterior horn of the lateral ventricle in adult mice allowed us to detect a population of previously unknown mono- and bipolar cells whose bodies and processes were coated with polymorphous spines (PS) (Morfologiya, 135, No. 3, 7-19 (2009)). CR-positive spiny (CR(+)PS) cells did not contain GAD67 and were located in the white matter and layers V-VI of the frontal area of the dorsomedial cortex close to the cingulum, the rostrodorsal part of the caudate-putamen, the anterior olfactory nucleus, and the subependyma of the dorsolateral angle of the lateral ventricle. We report here studies of the distribution of these cells in seven-day-old mice. Comparative topographic analysis of definitive and early CR(+)PS cells showed that in seven-day-old mice, CR(+)PS cells were absent from the sites at which they were seen in adults, i.e., the anterior olfactory cortex, the cortical plate, and the inner part of the neostriatum. In addition, small numbers of CR(+)PS-like cells were seen at this age within the dorsal migration pathway, at the anterior margin of the neostriatum, along the dorsal border of the neostriatum with the corpus callosum, in the subependymal layer of the lateral wall of the lateral ventricle, and in the cingulum area. These data demonstrate that CR(+)PS cells may have a postnatal origin. Experiments to verify this hypothesis were performed using postnatal administration of bromodeoxyuridine (BrdU) to mice aged 2-4 days, followed by assessment of brain sections fixed at age 20 days. Double immunolabeling of sections for CR and BrdU demonstrated the presence of CR(+)PS cells containing postnatally supplied BrdU. These data provide evidence that at least some CR(+)PS cells undergo mitosis at postnatal age. In all probability, during the period from 7 to 20 days of postnatal development, CR(+)PS cells migrate to the sites that they occupy in adult animals. PMID:20721693

  12. Improved function and proliferation of adult human beta cells engrafted in diabetic immunodeficient NOD-scid IL2rγnull mice treated with alogliptin

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    Jurczyk A

    2013-12-01

    Full Text Available Agata Jurczyk,1 Philip diIorio,1 Dean Brostowin,1 Linda Leehy,1 Chaoxing Yang,1 Fumihiko Urano,2 David M Harlan,3 Leonard D Shultz,4 Dale L Greiner,1 Rita Bortell1 1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, 2Department of Medicine, Washington University School of Medicine, St Louis, MO, 3Department of Medicine, University of Massachusetts Medical School, Worcester, MA, 4The Jackson Laboratory, Bar Harbor, ME, USA Purpose: Dipeptidyl-peptidase-4 (DPP-4 inhibitors are known to increase insulin secretion and beta cell proliferation in rodents. To investigate the effects on human beta cells in vivo, we utilize immunodeficient mice transplanted with human islets. The study goal was to determine the efficacy of alogliptin, a DPP-4 inhibitor, to enhance human beta cell function and proliferation in an in vivo context using diabetic immunodeficient mice engrafted with human pancreatic islets. Methods: Streptozotocin-induced diabetic NOD-scid IL2rγnull (NSG mice were transplanted with adult human islets in three separate trials. Transplanted mice were treated daily by gavage with alogliptin (30 mg/kg/day or vehicle control. Islet graft function was compared using glucose tolerance tests and non-fasting plasma levels of human insulin and C-peptide; beta cell proliferation was determined by bromodeoxyuridine (BrdU incorporation. Results: Glucose tolerance tests were significantly improved by alogliptin treatment for mice transplanted with islets from two of the three human islet donors. Islet-engrafted mice treated with alogliptin also had significantly higher plasma levels of human insulin and C-peptide compared to vehicle controls. The percentage of insulin+BrdU+ cells in human islet grafts from alogliptin-treated mice was approximately 10-fold more than from vehicle control mice, consistent with a significant increase in human beta cell proliferation. Conclusion: Human islet-engrafted immunodeficient mice

  13. Promotion versus suppression of rat colon carcinogenesis by chlorophyllin and chlorophyll: modulation of apoptosis, cell proliferation, and β-catenin/Tcf signaling

    International Nuclear Information System (INIS)

    The carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 1,2-dimethylhydrazine (DMH) induce colon tumors in the rat that contain mutations in β-catenin, but the mutation pattern can be influenced by exposure to dietary phytochemicals, such as the water-soluble derivative of chlorophyll called chlorophyllin. Whereas chlorophyllin is an effective blocking agent during the initiation phase, post-initiation responses depend upon the exposure protocol, and can be influenced by the initiating agent and the concentration of chlorophyllin. Post-initiation treatment with 0.001% chlorophyllin (w/v) in the drinking water promoted colon carcinogenesis in the rat, but much higher concentrations (1.0% chlorophyllin) led to suppression. Bromodeoxyuridine and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) indices revealed that the promotional concentration of 0.001% chlorophyllin increased the ratio of cell proliferation to apoptosis in the colonic crypts, whereas concentrations in the range 0.01-1.0% chlorophyllin modestly reduced this ratio. Molecular studies showed that the spectrum of β-catenin mutations was markedly different in chlorophyllin-promoted colon tumors--many of the mutations led to direct substitutions of critical Ser/Thr residues within the glycogen synthase kinase-3β (GSK-3β) region, whereas in all other groups, including DMH and IQ controls, the mutations typically affected amino acids adjacent to Ser33. Substitution of critical Ser/Thr residues caused β-catenin and c-Jun proteins to be markedly over-expressed compared with tumors in which the mutations substituted amino acid residues flanking these critical Ser/Thr sites. In a separate study, rats were exposed to IQ or azoxymethane (AOM), a metabolite of DMH, and they were treated post-initiation with chlorophyllin, chlorophyll, copper, or phytol in the diet. Natural chlorophyll (0.08%) suppressed AOM- and IQ-induced aberrant crypt foci (ACF), whereas chlorophyllin had no

  14. Inhibitory effects of idoxifene on hepatic fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    Ya-jun ZHOU; Dong-mei YIN; Hong-shan CHEN; Jian-hua SHI; Bao-xi SHA; Xing WANG

    2005-01-01

    Aim: To investigate the effects of a tissue-specific selective estrogen receptor modulator, idoxifene, on hepatic fibrosis in rats. Methods: Hepatic fibrosis was induced by dimethylnitrosamine (DMN) in male rats. The DMN model of hepatic fibrosis and the hepatocytes undergoing oxidative stress were treated with idoxifene respectively. The effect of idoxifene on hepatic fibrosis in the DMN model was examined by immunohistochemistry. Effects of idoxifene on antioxidant enzyme levels of copper, zinc-dependent superoxide dismutase (CuZn-SOD),and cellular glutathione peroxidase (GSHPx) were measured by ELISA. Effects of idoxifene on activation, proliferation, and apoptosis of culture-activated hepatic stellate cells (HSC) were analysed by immunohistochemistry, bromodeoxyuridine (BrdU) uptake, and flow cytometry, respectively. Results: Idoxifene could mark edly suppress DMN-induced hepatic fibrosis in male rats. A treatment of 0.4mg.kg-1.d-1 of idoxifene reduced the protein levels of collagen in the DMN model by 41.19% (P<0.05). Protein level of CuZn-SOD and activitiy of GSHPx in liver treated with DMN plus 0.4 mg.kg-1.d-1 of idoxifene were 2.65 times (P<0.05) and 2.08 times greater (P<0.05) than that of liver treated with DMN alone respectively.The protein level of CuZn-SOD and activity of GSHPx in cultured rat hepatocytes treated with ferric nitrilotriacetate (FeNTA) plus 1 × 10-7 mol/L of idoxifene were 3.43 times (P<0.05) and 2.52 times (P<0.05) greater than that treated with FeNTA alone. Idoxifene could inhibit HSC activation. Compared with the control, the uptake of BrdU in HSC cultured with 1× 10-7 mol/L of idoxifene was reduced by 51.87 % (P<0.05), and the number of apoptotic HSCs cultured with 1 × 10-7 mol/L of idoxifene increased by 94.52% (P<0.05). Conclusion: Idoxifene showed inhibitory action on hepatic fibrosis in male rats.

  15. The method validation step of biological dosimetry accreditation process

    International Nuclear Information System (INIS)

    One of the missions of the Laboratory of Biological Dosimetry (L.D.B.) of the Institute for Radiation and Nuclear Safety (I.R.S.N.) is to assess the radiological dose after an accidental overexposure suspicion to ionising radiation, by using radio-induced changes of some biological parameters. The 'gold standard' is the yield of dicentrics observed in patients lymphocytes, and this yield is converted in dose using dose effect relationships. This method is complementary to clinical and physical dosimetry, for medical team in charge of the patients. To obtain a formal recognition of its operational activity, the laboratory decided three years ago, to require an accreditation, by following the recommendations of both 17025 General Requirements for the Competence of Testing and Calibration Laboratories and 19238 Performance criteria for service laboratories performing biological dosimetry by cyto-genetics. Diagnostics, risks analysis were realized to control the whole analysis process leading to documents writing. Purchases, personnel department, vocational training were also included in the quality system. Audits were very helpful to improve the quality system. One specificity of this technique is that it is not normalized therefore apart from quality management aspects, several technical points needed some validations. An inventory of potentially influent factors was carried out. To estimate their real effect on the yield of dicentrics, a Placket-Burman experimental design was conducted. The effect of seven parameters was tested: the BUdr (bromodeoxyuridine), PHA (phytohemagglutinin) and colcemid concentration, the culture duration, the incubator temperature, the blood volume and the medium volume. The chosen values were calculated according to the uncertainties on the way they were measured i.e. pipettes, thermometers, test tubes. None of the factors has a significant impact on the yield of dicentrics. Therefore the uncertainty linked to their use was considered as

  16. The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano

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    Adamski Zbigniew

    2009-07-01

    Full Text Available Abstract Background Macrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts in S-phase. Results The tail plate accomodates the male genital apparatus and consists of about 3,100 cells, about half of which are epidermal cells. A distinct regeneration blastema, characterized by a local accumulation of rapidly proliferating neoblasts and consisting of about 420 cells (excluding epidermal cells, was formed 24 hours after amputation. Differentiated cells in the blastema were observed two days after amputation (with about 920 blastema cells, while the male genital apparatus required four to five days for full differentiation. At all time points, mitoses were found within the blastema. At the place of organ differentiation, neoblasts did not replicate or divide. After three days, the blastema was made of about 1420 cells and gradually transformed into organ primordia, while the proliferation rate decreased. The cell number of the tail plate, including about 960 epidermal cells, was restored to 75% at this time point. Conclusion Regeneration after artificial amputation of the tail plate of adult specimens of Macrostomum lignano involves wound healing and the formation of a regeneration blastema. Neoblasts undergo extensive proliferation within the blastema. Proliferation patterns of S-phase neoblasts indicate that neoblasts are either determined to follow a specific cell fate not before, but after going through S-phase, or that they can be redetermined after S-phase. In pulse-chase experiments, dispersed distribution of label suggests that S-phase labeled progenitor cells of the male genital apparatus undergo further proliferation before

  17. Control of Vascular Smooth Muscle Cell Growth by Connexin 43

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    Chintamani eJoshi

    2012-06-01

    Full Text Available Connexin 43 (Cx43, the principal gap junction protein in vascular smooth muscle cells (VSMCs, regulates movement of ions and other signaling molecules through gap junction intercellular communication (GJIC and plays important roles in maintaining normal vessel function; however, many of the signaling mechanisms controlling Cx43 in VSMCs are not clearly described. The goal of this study was to investigate mechanisms of Cx43 regulation with respect to VSMC proliferation. Treatment of rat primary VSMCs with the cAMP analog 8Br-cAMP, the soluble guanylate cyclase (sGC stimulator BAY 41-2272 (BAY, or the Cx inducer diallyl disulfide (DADS significantly reduced proliferation after 72 h compared to vehicle controls. Bromodeoxyuridine uptake revealed reduction (p<.001 in DNA synthesis after 6 h and flow cytometry showed reduced (40% S phase cell numbers after 16 h in DADS-treated cells compared to controls. Cx43 expression significantly increased after 270 min treatment with 8Br-cAMP, 8Br-cGMP, BAY or DADS. Inhibition of PKA, PKG or PKC reversed 8Br-cAMP-stimulated increases in Cx43 expression, whereas only PKG or PKC inhibition reversed 8Br-cGMP- and BAY-stimulated increases in total Cx43. Interestingly, stimulation of Cx43 expression by DADS was not dependent on PKA, PKG or PKC. Using fluorescence recovery after photobleaching, only 8Br-cAMP or DADS increased GJIC with 8Br-cAMP mediated by PKC and DADS mediated by PKG. Further, DADS significantly increased phosphorylation at the MAPK-sensitive serine (Ser255 and Ser279, the cell cycle regulatory kinase-sensitive Ser262 and the PKC-sensitive Ser368 after 30 min while 8Br-cAMP significantly increased phosphorylation only at Ser279 compared to controls. This study demonstrates that 8Br-cAMP- and DADS-enhanced GJIC rather than Cx43 expression and/or phosphorylation plays an important role in regulation of VSMC proliferation and provides new insights into the growth-regulatory capacities of Cx43 in VSMCs.

  18. Chronic treatment with fluoxetine for more than 6 weeks decreases neurogenesis in the subventricular zone of adult mice

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    Ohira Koji

    2011-03-01

    Full Text Available Abstract Background Recent studies indicate that chronic treatment with serotonergic antidepressants upregulates adult neurogenesis of the dentate gyrus (DG. In contrast, some studies claimed that there was very little alteration of neurogenesis in the subventricular zone (SVZ by the antidepressants. Since almost all of those studies treated animals with drugs for 2 to 4 weeks as chronic treatment models of antidepressants, it is possible that antidepressant treatments for longer periods would affect adult neurogenesis in the SVZ. Results In the present study, we examined the effects of long-term (up to 9 weeks administration of fluoxetine (FLX, a selective serotonin reuptake inhibitor, on cell proliferation and survival in the DG and the SVZ of adult mice. As reported previously, in the DG of mice treated with FLX for 3, 6, or 9 weeks that were also injected with 5-bromodeoxyuridine (BrdU in the last 3 days before perfusion, the numbers of Ki67- and BrdU-positive cells, which are cell proliferation markers, were significantly upregulated even at 3 weeks after the onset of the FLX treatments, and these increases were sustained in mice treated with FLX for 9 weeks. On the other hand, in the SVZ, we found a small, insignificant decrease in the numbers of Ki67- and BrdU-positive cells at 3 weeks, followed by highly significant decreases in the numbers of Ki67- and BrdU-positive cells at both 6 and 9 weeks. Furthermore, among olfactory newly generated cells that survived for 3 weeks after BrdU injection, the number of new cells was decreased at 9 weeks of FLX treatment. Conclusions These results demonstrate that long-term (more than 6 weeks treatment with FLX has the opposite effect on neurogenesis in the SVZ than it does in the DG. The results also suggest that the decrease in neurogenesis in the SVZ might be involved in some aspects of the drugs' therapeutic effects on depression. In addition, our findings raise the possibility that some of the

  19. A phase 3 randomized study of radiotherapy plus procarbazine, CCNU, and vincristine (PCV) with or without BUdR for the treatment of anaplastic astrocytoma: a preliminary report of RTOG 9404

    International Nuclear Information System (INIS)

    Purpose: This study was an open label, randomized Phase 3 trial in newly diagnosed patients with anaplastic glioma comparing radiotherapy plus adjuvant procarbazine, CCNU, and vincristine (PCV) chemotherapy with or without bromodeoxyuridine (BUdR) given as a 96-hour infusion each week of radiotherapy. Methods and Materials: Only patients 18 years or older with newly diagnosed anaplastic glioma were eligible; central pathology review was accomplished, but was not mandated prior to registration. The study had initially opened as a Northern California Oncology Group (NCOG) trial in 1991, becoming an Intergroup RTOG, SWOG, and NCCTG study in July 1994. Total accrual of 293 patients was planned as the sample size, using survival and time to tumor progression as the primary endpoints. The experiment arm (RT/BUdR plus PCV) was to be compared to the control arm (RT plus PCV) using an alpha = 0.05, one-tailed, with a power of 85% for detecting an increase in median survival from 160 to 240 weeks, assuming a 3-year follow-up after completion of enrollment. Results: As of July 1996, 281 patients had been randomized; 53 (20%) were ineligible, primarily based upon central pathology review, and another 39 cases were canceled. In total, 30% of cases were excluded from analysis. The treatment arms were well balanced despite this rate of exclusion. The RTOG Data Monitoring Committee recommended suspension of enrollment in July 1996 based upon a stochastic curtailment analysis which strongly suggested that the addition of BUdR would not be associated with increased survival. In February 1997, the study was closed prior to full enrollment. At that time, the 1-year survival estimates were 82% versus 68% for RT plus PCV and RT/BUdR plus PCV respectively (one-sided, p = 0.96). The conditional power analysis indicated that even with an additional 12 months of additional accrual and follow-up the probability of detecting the prespecified difference was less than 0.01%. The differences in

  20. LXA4 actions direct fibroblast function and wound closure

    International Nuclear Information System (INIS)

    Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA4 receptor (ALX/FPR2

  1. Similarity between the effects of carbon-ion irradiation and X-irradiation on the development of rat brain

    International Nuclear Information System (INIS)

    The effects of carbon-ion irradiation and X-irradiation on the development of rat brain were compared. Twenty pregnant rats were injected with bromodeoxyuridine (BrdU) at 9 pm on day 18 pregnancy and divided into five groups. Three hours after injection (day 19.0) one group was exposed to 290 MeV/u carbon-ion radiation by a single dose of 1.5 Gy. Other groups were exposed to X-radiation by 1.5, 2.0 or 2.5 Gy, or sham-treated, respectively. Fetuses were removed from one dam in each group 8 h after exposure and examined histologically. Extensive cell death was observed in the brain mantle from the irradiated groups. The cell death after 1.5 Gy carbon-ion irradiation was remarkably more extensive than that after 1.5 Gy X-irradiation, but comparable to that after 2.0 Gy or 2.5 Gy X-irradiation. The remaining rats were allowed to give birth and the offspring were sacrificed at 6 weeks of age. All of the irradiated offspring manifested microcephaly. The size of the brain mantle exposed to 1.5 Gy carbon-ion radiation was significantly smaller than that exposed to 1.5 Gy X-radiation and larger than that exposed to 2.5 Gy X-radiation. A histological examination of the cerebral cortex revealed that cortical layers II-IV were malformed. The defect by 1.5 Gy carbon-ion irradiation was more severe than that by the same dose of X-irradiation. Although the BrdU-incorporated neurons were greatly reduced in number in all irradiated groups, these cells reached the superficial area of the cortex. These findings indicated that the effects of both carbon-ion irradiation and X-irradiation on the development of rat brain are similar in character, and the effect of 1.5 Gy carbon-ion irradiation compares to that of 2.0-2.5 Gy X-irradiation. (author)

  2. The bradykinin B2 receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines

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    Kramarenko II

    2012-07-01

    Full Text Available Inga I Kramarenko1, Thomas A Morinelli1,2, Marlene A Bunni1,2, John R Raymond Sr3, Maria N Garnovskaya11Department of Medicine (Nephrology Division, Medical University of South Carolina, Charleston, SC, USA; 2Medical and Research Services of the Ralph H Johnson Veterans Affairs Medical Center, Charleston, SC, USA; 3Medical College of Wisconsin, Milwaukee, WI, USABackground: The vasoactive peptide bradykinin (BK acts as a potent growth factor for normal kidney cells, but there have been few studies on the role of BK in renal cell carcinomas.Purpose: In this study, we tested the hypothesis that BK also acts as a mitogen in kidney carcinomas, and explored the effects of BK in human renal carcinoma A498 cells.Methods: The presence of mRNAs for BK B1 and BK B2 receptors in A498 cells was demonstrated by reverse transcription–polymerase chain reaction. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE with a Cytosensor microphysiometer, and assessed extracellular signal-regulated kinase (ERK activation by Western blotting.Results: Exposure to 100 nM of BK resulted in the rapid elevation of intracellular Ca2+, caused a ≥30% increase in NHE activity, and a ≥300% increase in ERK phosphorylation. All BK signals were blocked by HOE140, a BK B2 receptor antagonist, but not by a B1 receptor antagonist. Inhibitor studies suggest that BK-induced ERK activation requires phospholipase C and protein kinase C activities, and is Ca2+/calmodulin-dependent. The amiloride analog 5-(N-methyl-N-isobutyl-amiloride (MIA blocked short-term NHE activation and inhibited ERK phosphorylation, suggesting that NHE is critical for ERK activation by BK. BK induced an approximately 40% increase in the proliferation of A498 cells as assessed by bromodeoxyuridine uptake. This effect was blocked by the ERK inhibitor PD98059, and was dependent on NHE activity

  3. Relationship of the demethylation of the DNA with the induction of the sister chromatid exchanges (SCE) In vivo; Relacion de la desmetilacion del ADN con la induccion de intercambios en las cromatidas hermanas (ICH) In vivo

    Energy Technology Data Exchange (ETDEWEB)

    Toribio E, E

    2005-07-01

    The methylation of the DNA is an epigenetic modification that has an important paper in the regulation of the functionality of the genome of the organisms. It can be altered by demethylation processes, either natural or experimentally induced. The 5-azacytidine (Aza) is a compound that causes the demethylation of the DNA (dm-DNA), inducing with it, expression genic and increase in the frequency of the Sister Chromatid Exchange (SCE). The SCE is a genotoxicity indicator, caused by diverse mutagens and carcinogen. Since the biological meaning and the formation mechanism of this phenomenon has not been totally illustrious, the exploration of the relation between the dm-DNA and the induction of SCE, it could offer new knowledge to explain those queries. The purpose of this work was to study in cells of the mouse bone marrow In vivo, the effect of the Aza on the induction of SCE, based on two aspects: 1) dose answer and 2) the effectiveness of multiple exhibition. To six groups of three to five animals, they are administered Aza to dose of 5, 10, 15 or 20 mg/Kg of weight; in sharp or multiple form, previously to the bromodeoxyuridine supply and 24 h was sacrificed after this; 2 h after an injection with colchicine. Preparations of those metaphases were made, those which were dyed by means of a technique of fluorescence more Giemsa. It was observed that to sharp low dose, the Aza produced an increment in the frequency of SCE that although small it was proportional and statistically significant. To sharp and multiple high doses, the Aza doesn't cause additional increments of SCE, but if toxicity at cellular level and of individuals. It is concluded that a relationship exists between the dm-DNA and the induction of SCE. It is suggested that the total demethylation of the DNA causes 2 SCE/Cell in cells of the mouse bone marrow, or that the cytotoxicity prevents to evidence a bigger induction. (Author)

  4. Chromogranin A and its fragments as regulators of small intestinal neuroendocrine neoplasm proliferation.

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    Francesco Giovinazzo

    Full Text Available INTRODUCTION: Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation. METHODS: Chromogranin A mRNA (PCR and protein (ELISA/western blot were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay, proliferation (bromodeoxyuridine ELISA and expression of AKT/AKT-P (CASE ELISA/western blot in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines. RESULTS: Chromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001 in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05. This inhibition also decreased secretion of chromogranin A (p<0.05 and 5-HT (p<0.05 as well as expression of vasostatin. Metastatic small intestinal NEN cell lines were stimulated (50-80%, p<0.05 and AKT phosphorylated (Ser473: p<0.05 by vasostatin I, which was completely reversed by RAD001 (p<0.01 and AKT antisense (p<0.05 while chromostatin inhibited proliferation (~50%, p<0.05. CONCLUSION: Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of

  5. cGMP modulates stem cells differentiation to neurons in brain in vivo.

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    Gómez-Pinedo, U; Rodrigo, R; Cauli, O; Herraiz, S; Garcia-Verdugo, J-M; Pellicer, B; Pellicer, A; Felipo, V

    2010-02-17

    During brain development neural stem cells may differentiate to neurons or to other cell types. The aim of this work was to assess the role of cGMP (cyclic GMP) in the modulation of differentiation of neural stem cells to neurons or non-neuronal cells. cGMP in brain of fetuses was reduced to 46% of controls by treating pregnant rats with nitroarginine-methylester (L-NAME) and was restored by co-treatment with sildenafil.Reducing cGMP during brain development leads to reduced differentiation of stem cells to neurons and increased differentiation to non-neuronal cells. The number of neurons in the prefrontal cortex originated from stem cells proliferating on gestational day 14 was 715+/-14/mm(2) in control rats and was reduced to 440+/-29/mm(2) (61% of control) in rats treated with L-NAME. In rats exposed to L-NAME plus sildenafil, differentiation to neurons was completely normalized, reaching 683+/-11 neurons/mm(2). In rats exposed to sildenafil alone the number of cells labelled with bromodeoxyuridine (BrdU) and NeuN was 841+/-16/mm(2). In prefrontal cortex of control rats 48% of the neural stem cells proliferating in gestational day 14 differentiate to neurons, but only 24% in rats exposed to L-NAME. This was corrected by sildenafil, 40% of cells differentiate to neurons. Similar results were obtained for neurons proliferating during all developmental period. Treatment with L-NAME did not reduce the total number of cells labelled with BrdU, further supporting that L-NAME reduces selectively the differentiation of stem cells to neurons. Similar results were obtained in hippocampus. Treatment with L-NAME reduced the differentiation of neural stem cells to neurons, although the effect was milder than in prefrontal cortex. These results support that cGMP modulates the fate of neural stem cells in brain in vivo and suggest that high cGMP levels promote its differentiation to neurons while reduced cGMP levels promote differentiation to non-neuronal cells. PMID:19958812

  6. Transcranial low-level laser therapy improves neurological performance in traumatic brain injury in mice: effect of treatment repetition regimen.

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    Weijun Xuan

    Full Text Available Low-level laser (light therapy (LLLT has been clinically applied around the world for a spectrum of disorders requiring healing, regeneration and prevention of tissue death. One area that is attracting growing interest in this scope is the use of transcranial LLLT to treat stroke and traumatic brain injury (TBI. We developed a mouse model of severe TBI induced by controlled cortical impact and explored the effect of different treatment schedules. Adult male BALB/c mice were divided into 3 broad groups (a sham-TBI sham-treatment, (b real-TBI sham-treatment, and (c real-TBI active-treatment. Mice received active-treatment (transcranial LLLT by continuous wave 810 nm laser, 25 mW/cm(2, 18 J/cm(2, spot diameter 1 cm while sham-treatment was immobilization only, delivered either as a single treatment at 4 hours post TBI, as 3 daily treatments commencing at 4 hours post TBI or as 14 daily treatments. Mice were sacrificed at 0, 4, 7, 14 and 28 days post-TBI for histology or histomorphometry, and injected with bromodeoxyuridine (BrdU at days 21-27 to allow identification of proliferating cells. Mice with severe TBI treated with 1-laser Tx (and to a greater extent 3-laser Tx had significant improvements in neurological severity score (NSS, and wire-grip and motion test (WGMT. However 14-laser Tx provided no benefit over TBI-sham control. Mice receiving 1- and 3-laser Tx had smaller lesion size at 28-days (although the size increased over 4 weeks in all TBI-groups and less Fluoro-Jade staining for degenerating neurons (at 14 days than in TBI control and 14-laser Tx groups. There were more BrdU-positive cells in the lesion in 1- and 3-laser groups suggesting LLLT may increase neurogenesis. Transcranial NIR laser may provide benefit in cases of acute TBI provided the optimum treatment regimen is employed.

  7. The Success of Thread-embedding Therapy in Generating Hair Re-growth in Mice Points to Its Possibly Having a Similar Effect in Humans

    Science.gov (United States)

    Shin, Hyun Jong; Lee, Dong-Jin; Kwon, Kang; Seo, Hyung-Sik; Jeong, Han-Sol; Lee, Ji-Yeon; Ha, Ki-Tae; Lee, Chang-Hyun; Jang, Yong-Suk; Lee, Byung-Wook; Kim, Byung Joo; Jung, Myeong-Ho

    2015-01-01

    Objectives: Recently, thread-embedding therapy (TET) has been widely applied in Korean medicine for cosmetic purposes such as reducing skin wrinkles. An inserted thread was reported to have induced continuous stimulation, followed by support for connective tissue regeneration. However, the potential role of TET in hairgrowth has not yet been reported. Methods: We designed this study to evaluate whether TET has a hair-growth-promoting effect. C57 black 6 (C57BL/6) mice were divided into three groups: normal saline-treated, minoxidil-treated, and thread-embedded groups. Normal saline or 5% minoxidil was topically sprayed on the dorsal skin of the mice once a day for 16 days. Medical threads were embedded into the dorsal skin of the mice in a single application. Hair growth activity was evaluated by using dermoscopic and microscopic observations. Sections of the dorsal skin were stained with hematoxylin and eosin. Expressions of bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7), and fibroblast growth factor-5 (FGF-5) were detected by using immunohistochemical staining. A reverse transcription-polymerase chain reaction (RT-PCR) analysis was adopted to measure the messenger RNA (mRNA) expressions of FGF-7 and FGF-5. Results: TET enhanced anagen development in the hair follicles of C57BL/6 mice. The expressions of BrdU and PCNA, both of which imply active cellular proliferation, were increased by using TET. Moreover, TET increased the expression of FGF-7, an anagen-inducing growth factor, while decreasing the expression of FGF-5, an anagen-cessation growth factor, both at the protein and the mRNA levels. Conclusion: TET enhanced hair re-growth in C57BL/6 mice. TET regulated the expressions of anagen-associated growth factors and activated the proliferation of hair follicular cells in depilated skin lesions. Considering its long-lasting effect, TET may be a good alternative therapeutic for the treatment of alopecia

  8. The new nano-complex, Hep-c, improves the immunogenicity of the hepatitis B vaccine.

    Science.gov (United States)

    Fakharzadeh, Saideh; Kalanaky, Somayeh; Hafizi, Maryam; Goya, Mohammad Mahdi; Masoumi, Zahra; Namaki, Said; Shakeri, Nezhat; Abbasi, Maryam; Mahdavi, Mehdi; Nazaran, Mohammad Hassan

    2013-05-24

    Prevention of hepatitis B requires a vaccine that stimulates the humoral and cellular immune responses in a balanced manner, particularly those associated with Th1 and cytotoxic T cells. Alum adjuvant is currently used in the hepatitis B vaccine formulations but it lacks the efficiency of establishing such immune responses. Therefore, for accessing a suitable vaccine to prevent this fatal disease, it is essential to design and construct a new adjuvant which can overcome the limitations of the alum adjuvant and can stimulate a strong Th1 response as used along with it. In the present study, the adjuvant effect of Hep-c, the first nano-complex which was synthesized by nanochelating technology to improve the immunogenicity of the vaccine against hepatitis B, had been evaluated. Female Balb/c mice were divided into 7 groups and were injected with 10μg/ml of the hepatitis B vaccine and different doses of Hep-c for 3 times. Groups merely treated with the vaccine, Hep-c or phosphate buffered solution were used as control. Total specific antibody, IgG1, IgG2a, IgG2b, IgM, interleukin-4 (IL-4) and interferon-gamma (IFN-γ) levels were examined by the ELISA method. The proliferative response of the splenocytes was evaluated using bromodeoxyuridine assay. Results showed that immunization with hepatitis B vaccine and Hep-c increased the lymphocyte proliferation and specific IgM and IgG2a compared to the hepatitis B vaccine immunized group. Also, this nano-complex significantly increased the IFN-γ and IL-4 cytokine levels compared to the hepatitis B vaccine immunized group. Our findings show that Hep-c can not only preserve the alum capacity to effectively stimulate production of the antibodies but also cover its inefficiency in inducing Th1 response and prompting cellular immunity. Thus, by boosting the performance of the hepatitis B vaccine, it seemed that this nano-adjuvant has the suitable potential to be used in the commercial HBS vaccine formulation. PMID:23583463

  9. A novel human ex vivo model for the analysis of molecular events during lung cancer chemotherapy

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    Lang Dagmar S

    2007-06-01

    Full Text Available Abstract Background Non-small cell lung cancer (NSCLC causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC. Methods In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine uptake as markers for proliferation and of cleaved (activated effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines. Results Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC and human cell lines (CPC-N, HEK proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC. Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas. Conclusion Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST

  10. DNA replication kinetics in x-irradiated Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    The kinetics of semiconservative DNA replication have been studied in both asynchronous and synchronized Chinese hamster ovary cells (CHO) irradiated with x-ray doses up to 3000 rad. Amounts of DNA replicated were determined by isopycnic gradient centrifugation of DNA from cells which were incubated after irradiation in medium containing 5-bromodeoxyuridine (50 μg/ml) and 5-fluorodeoxyuridine (0.1 μg/ml). The results confirm that cells irradiated in early G1 phase experience a delay in their entry into S phase. This G1 block is dose independent in the range from 300 to 3000 rad and is 0.5 to 0.7 hr in length. Cells at the G1/S boundary are insensitive to x-ray induced perturbations of bulk DNA synthetic rates when exposed to doses less than 1000 rad. At doses in excess of 1000 rad, these cells are inhibited from replicating their DNA for a time, but ultimately replicate near-control levels of their DNA. Cells irradiated in S phase again show no effects of x-ray doses below 1000 rad on their ability to replicate bulk DNA. After a 3000-rad exposure, however, the rate of DNA replication in these S-phase cells is markedly reduced compared to that of controls. Irradiation of asynchronous cells with doses from 150 to 3000 rad does reduce the rate of semiconservative DNA replication in these cultures in a dose-dependent manner. These results confirm that x-ray doses greater than 1000 rad reduce the rate of DNA synthesis in irradiated S-phase cells, thus prolonging the length of S phase. The combined data from asynchronous or synchronized cultures, irradiated with x-ray doses less than 1000 rad, indicate that at least a portion of the reduction in DNA replication rates in irradiated asynchronous CHO cultures is due to the x-ray induced G1 block, which reduces the overall number of cells in S phase after irradiation

  11. CCL2/MCP-1 modulation of microglial activation and proliferation

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    Garcia-Bueno Borja

    2011-07-01

    Full Text Available Abstract Background Monocyte chemoattractant protein (CCL2/MCP-1 is a chemokine that attracts cells involved in the immune/inflammatory response. As microglia are one of the main cell types sustaining inflammation in brain, we proposed here to analyze the direct effects of MCP-1 on cultured primary microglia. Methods Primary microglia and neuronal cultures were obtained from neonatal and embryonic Wistar rats, respectively. Microglia were incubated with different concentrations of recombinant MCP-1 and LPS. Cell proliferation was quantified by measuring incorporation of bromodeoxyuridine (BrdU. Nitrite accumulation was measured using the Griess assay. The expression and synthesis of different proteins was measured by RT-PCR and ELISA. Cell death was quantified by measuring release of LDH into the culture medium. Results MCP-1 treatment (50 ng/ml, 24 h did not induce morphological changes in microglial cultures. Protein and mRNA levels of different cytokines were measured, showing that MCP-1 was not able to induce proinflammatory cytokines (IL-1β, IL6, MIP-1α, either by itself or in combination with LPS. A similar lack of effect was observed when measuring inducible nitric oxide synthase (NOS2 expression or accumulation of nitrites in the culture media as a different indicator of microglial activation. MCP-1 was also unable to alter the expression of different trophic factors that were reduced by LPS treatment. In order to explore the possible release of other products by microglia and their potential neurotoxicity, neurons were co-cultured with microglia: no death of neurons could be detected when treated with MCP-1. However, the presence of MCP-1 induced proliferation of microglia, an effect opposite to that observed with LPS. Conclusion These data indicate that, while causing migration and proliferation of microglia, MCP-1 does not appear to directly activate an inflammatory response in this cell type, and therefore, other factors may be

  12. The Success of Thread-embedding Therapy in Generating Hair Re-growth in Mice Points to Its Possibly Having a Similar Effect in Humans

    Directory of Open Access Journals (Sweden)

    Hyun Jong Shin

    2015-12-01

    Full Text Available Objectives: Recently, thread-embedding therapy (TET has been widely applied in Korean medicine for cosmetic purposes such as reducing skin wrinkles. An inserted thread was reported to have induced continuous stimulation, followed by support for connective tissue regeneration. However, the potential role of TET in hairgrowth has not yet been reported. Methods: We designed this study to evaluate whether TET has a hair-growth-promoting effect. C57 black 6 (C57BL/6 mice were divided into three groups: normal saline-treated, minoxidil-treated, and thread-embedded groups. Normal saline or 5% minoxidil was topically sprayed on the dorsal skin of the mice once a day for 16 days. Medical threads were embedded into the dorsal skin of the mice in a single application. Hair growth activity was evaluated by using dermoscopic and microscopic observations. Sections of the dorsal skin were stained with hematoxylin and eosin. Expressions of bromodeoxyuridine (BrdU, proliferating cell nuclear antigen (PCNA, fibroblast growth factor-7 (FGF-7, and fibroblast growth factor-5 (FGF-5 were detected by using immunohistochemical staining. A reverse transcription-polymerase chain reaction (RT-PCR analysis was adopted to measure the messenger RNA (mRNA expressions of FGF-7 and FGF-5. Results: TET enhanced anagen development in the hair follicles of C57BL/6 mice. The expressions of BrdU and PCNA, both of which imply active cellular proliferation, were increased by using TET. Moreover, TET increased the expression of FGF-7, an anagen-inducing growth factor, while decreasing the expression of FGF-5, an anagen-cessation growth factor, both at the protein and the mRNA levels. Conclusion: TET enhanced hair re-growth in C57BL/6 mice. TET regulated the expressions of anagen-associated growth factors and activated the proliferation of hair follicular cells in depilated skin lesions. Considering its long-lasting effect, TET may be a good alternative therapeutic for the treatment of

  13. Phenotype change and migration of adventitial fibroblasts during postangioplasty

    International Nuclear Information System (INIS)

    Objective: To verify fibroblasts translocation from adventitia into neointima by labeling adventitia cells with bromodeoxyuridine (BrDU) after angioplasty, and to explore the relationship of adventitial fibroblast with restenosis. Methods: Vascular restenosis model was created by injured intima of common carotid artery (CCA) of mouse with guide wire, adventitial fibroblasts were labeled with BrDU, and dynamic distribution of myofibroblasts in adventitia, media and neoitima was observed at different times (3 d, 7 d, 14 d and 28 d) by means of single/double-label immunohistochemistry, light microscope, electronic microscope and image analysis system. Results: 1.Immunohistochemistry: More adventitial fibroblasts combined with BrDU could be found in adventitia on the 3rd day of postangioplasty, and the number of this kind of cells reached the peak on 7th day, and at the same time fibroblasts changed their phenotypes and became myofibroblasts, which produced α-actin and extracellular matrix (ECM). On 14th day, the number of the positive cells decreased in adventitia, increased in media and neointima associated with intima thickening; on 28th day, while the number of fibroblasts labeled by BrDU returned to the basic-line in adventitia, media and intima, nevertheless, intima thickening and vascular stenosis and intimal ELM precipitation were still present. There were significant differences in the number of fibroblasts labeled with BrDU located in three layers of artery (P<0.05). 2. Electronic microscope: After angioplasty, the plasm of fibroblasts became rich, mitochondrious and increase of Golgi apparatus; and the amount of rough endoplasmic reticulums rose with more secretory granules, together with a great amount of collagen synthesized forming the microfilaments; on days of 7th and 14th, the wide pseudopodia of myofibroblasts could be found extending into the windows on the external elastic lamina (ELL) and the internal elastic lamina (ILL); and showing the tendency

  14. Hypoxia-Inducible Factor Pathway Inhibition Resolves Tumor Hypoxia and Improves Local Tumor Control After Single-Dose Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Helbig, Linda [OncoRay–National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden (Germany); Department of Radiation Oncology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden (Germany); Koi, Lydia [OncoRay–National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden (Germany); Department of Radiation Oncology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden (Germany); Deutsches Konsortium für Translationale Krebsforschung, Site Dresden, Dresden (Germany); Brüchner, Kerstin [Department of Radiation Oncology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden (Germany); Institute of Radiooncology Helmholtz-Zentrum Dresden-Rossendorf, Dresden (Germany); Gurtner, Kristin [Department of Radiation Oncology, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden (Germany); Hess-Stumpp, Holger; Unterschemmann, Kerstin [Global Drug Discovery, Bayer Pharma, Berlin (Germany); Pruschy, Martin [Radiation Oncology, University of Zurich, Zurich (Switzerland); and others

    2014-01-01

    Purpose: To study the effects of BAY-84-7296, a novel orally bioavailable inhibitor of mitochondrial complex I and hypoxia-inducible factor 1 (HIF-1) activity, on hypoxia, microenvironment, and radiation response of tumors. Methods and Materials: UT-SCC-5 and UT-SCC-14 human squamous cell carcinomas were transplanted subcutaneously in nude mice. When tumors reached 4 mm in diameter BAY-84-7296 (Bayer Pharma AG) or carrier was daily administered to the animals. At 7 mm tumors were either excised for Western blot and immunohistologic investigations or were irradiated with single doses. After irradiation animals were randomized to receive BAY-84-7296 maintenance or carrier. Local tumor control was evaluated 150 days after irradiation, and the dose to control 50% of tumors (TCD{sub 50}) was calculated. Results: BAY-84-7296 decreased nuclear HIF-1α expression. Daily administration of inhibitor for approximately 2 weeks resulted in a marked decrease of pimonidazole hypoxic fraction in UT-SCC-5 (0.5% vs 21%, P<.0001) and in UT-SCC-14 (0.3% vs 19%, P<.0001). This decrease was accompanied by a significant increase in fraction of perfused vessels in UT-SCC-14 but not in UT-SCC-5. Bromodeoxyuridine and Ki67 labeling indices were significantly reduced only in UT-SCC-5. No significant changes were observed in vascular area or necrosis. BAY-84-7296 before single-dose irradiation significantly decreased TCD{sub 50}, with an enhancement ratio of 1.37 (95% confidence interval [CI] 1.13-1.72) in UT-SCC-5 and of 1.55 (95% CI 1.26-1.94) in UT-SCC-14. BAY-84-7296 maintenance after irradiation did not further decrease TCD{sub 50}. Conclusions: BAY-84-7296 resulted in a marked decrease in tumor hypoxia and substantially reduced radioresistance of tumor cells with the capacity to cause a local recurrence after irradiation. The data suggest that reduction of cellular hypoxia tolerance by BAY-84-7296 may represent the primary biological mechanism underlying the observed enhancement of

  15. Similarity between the effects of carbon-ion irradiation and X-irradiation on the development of rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Inouye, Minoru; Hayasaka, Shizu; Murata, Yoshiharu [Nagoya Univ. (Japan). Research Inst. of Environmental Medicine; Takahashi, Sentaro; Kubota, Yoshihisa

    2000-09-01

    The effects of carbon-ion irradiation and X-irradiation on the development of rat brain were compared. Twenty pregnant rats were injected with bromodeoxyuridine (BrdU) at 9 pm on day 18 pregnancy and divided into five groups. Three hours after injection (day 19.0) one group was exposed to 290 MeV/u carbon-ion radiation by a single dose of 1.5 Gy. Other groups were exposed to X-radiation by 1.5, 2.0 or 2.5 Gy, or sham-treated, respectively. Fetuses were removed from one dam in each group 8 h after exposure and examined histologically. Extensive cell death was observed in the brain mantle from the irradiated groups. The cell death after 1.5 Gy carbon-ion irradiation was remarkably more extensive than that after 1.5 Gy X-irradiation, but comparable to that after 2.0 Gy or 2.5 Gy X-irradiation. The remaining rats were allowed to give birth and the offspring were sacrificed at 6 weeks of age. All of the irradiated offspring manifested microcephaly. The size of the brain mantle exposed to 1.5 Gy carbon-ion radiation was significantly smaller than that exposed to 1.5 Gy X-radiation and larger than that exposed to 2.5 Gy X-radiation. A histological examination of the cerebral cortex revealed that cortical layers II-IV were malformed. The defect by 1.5 Gy carbon-ion irradiation was more severe than that by the same dose of X-irradiation. Although the BrdU-incorporated neurons were greatly reduced in number in all irradiated groups, these cells reached the superficial area of the cortex. These findings indicated that the effects of both carbon-ion irradiation and X-irradiation on the development of rat brain are similar in character, and the effect of 1.5 Gy carbon-ion irradiation compares to that of 2.0-2.5 Gy X-irradiation. (author)

  16. Radiation and/or hyperthermia sensitivity of human melanoma cells after several days of incubation in media lacking serum or certain serum components

    International Nuclear Information System (INIS)

    Purpose: Complete serum and single growth factors have been reported to protect cells against the effects of hyperthermia. The phenomenon is not very well understood, especially with regard to the relative importance of the various serum components. There is also a need to clarify the possible involvement of changes in proliferation. The influence of serum and growth factors on radiation sensitivity has not been studied in detail. The present communication is an attempt to at least partly fill these gaps. Cells were exposed to X-rays and/or heat after incubation in sufficiently supplemented medium and media lacking certain serum components. Differences in clonogenic survival were recorded and related to the proliferative status under the various conditions. Methods and Materials: Human melanoma cells (MeWo) were used throughout. Cells were incubated for 3 or 6 days (a) in Ham's F12 medium with 20% fetal calf serum; (b) in medium without serum, but supplemented with the following components: insulin, transferrin, Na-selenite, and a lipid-protein complex; (c) to (f) in media lacking one or the other of these components; (g) in unsupplemented medium. Colony formation ability served as a measure of cell survival after exposure to radiation (250 kV X-rays) and/or hyperthermia (heating at 43 deg. C). In parallel, cell proliferation was characterized by the bromodeoxyuridine (BrdU) labeling index and the Ki-67 growth fraction, both determined by two-parameter flow cytometry. Results and Conclusions: Sensitivity was hardly influenced by 3 days of incubation in medium containing no supplement at all, whereas after 6 days under the same conditions survival curves both of irradiated and hyperthermia-treated cells had a strongly reduced shoulder or were considerably steeper. Experiments with media lacking only one or the other serum component, showed that deprivation of insulin, transferrin, and sodium selenite led to no more than a modest increase in cell killing, whereas the

  17. Single-Walled Carbon Nanotube (SWCNT-induced interstitial fibrosis in the lungs of rats is associated with increased levels of PDGF mRNA and the formation of unique intercellular carbon structures that bridge alveolar macrophages In Situ

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    Bermudez Edilberto

    2006-11-01

    Full Text Available Abstract Background Nanotechnology is a rapidly advancing industry with many new products already available to the public. Therefore, it is essential to gain an understanding of the possible health risks associated with exposure to nanomaterials and to identify biomarkers of exposure. In this study, we investigated the fibrogenic potential of SWCNT synthesized by chemical vapor deposition using cobalt (Co and molybdenum (Mo as catalysts. Following a single oropharyngeal aspiration of SWCNT in rats, we evaluated lung histopathology, cell proliferation, and growth factor mRNAs at 1 and 21 days post-exposure. Comparisons were made to vehicle alone (saline containing a biocompatible nonionic surfactant, inert carbon black (CB nanoparticles, or vanadium pentoxide (V2O5 as a known inducer of fibrosis. Results SWCNT or CB caused no overt inflammatory response at 1 or 21 days post-exposure as determined by histopathology and evaluation of cells (>95% macrophages in bronchoalveolar lavage (BAL fluid. However, SWCNT induced the formation of small, focal interstitial fibrotic lesions within the alveolar region of the lung at 21 days. A small fraction of alveolar macrophages harvested by BAL from the lungs of SWCNT-exposed rats at 21 days were bridged by unique intercellular carbon structures that extended into the cytoplasm of each macrophage. These "carbon bridge" structures between macrophages were also observed in situ in the lungs of SWCNT-exposed rats. No carbon bridges were observed in CB-exposed rats. SWCNT caused cell proliferation only at sites of fibrotic lesion formation as measured by bromodeoxyuridine uptake into alveolar cells. SWCNT increased platelet-derived growth factor (PDGF-A, PDGF-B, and PDGF-C mRNA levels significantly at 1 day as measured by Taqman quantitative real-time RT-PCR. At 21 days, SWCNT did not increase any mRNAs evaluated, while V2O5 significantly increased mRNAs encoding PDGF-A, -B, and -C chains, PDGF-Rα, osteopontin

  18. Testicular involution prior to sex change in gilthead seabream is characterized by a decrease in DMRT1 gene expression and by massive leukocyte infiltration

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    Meseguer José

    2007-06-01

    Full Text Available Abstract Background Leukocytes are found within the testis of most, if not all, mammals and are involved in immunological surveillance, physiological regulation and tissue remodelling. The testis of seasonal breeding fish undergoes a regression process. In the present study, the second reproductive cycle (RC of the protandrous seasonal teleost fish, gilthead seabream, was investigated and the presence of leukocytes analysed. Special attention has been paid to the testicular degenerative process which is particularly active in the last stage of the second RC probably due to the immediacy of the sex change process. Methods Sexually mature specimens (n = 10–18 fish/month were sampled during the second RC. Some specimens were intraperitoneally injected with bromodeoxyuridin (BrdU before sampling. Light and electron microscopy was used to determine the different stages of gonadal development and the presence of leukocytes and PCR was used to analyse the gene expression of a testis-differentiating gene and of specific markers for macrophages and B and T lymphocytes. Immunocytochemistry and flow cytometry were performed using a specific antibody against acidophilic granulocytes from the gilthead seabream. Cell proliferation was detected by immunocytochemistry using an anti-BrdU antibody and apoptotic cells by in situ detection of DNA fragmentation. Results The fish in the western Mediterranean area developed as males during the first two RCs. The testis of all the specimens during the second RC underwent a degenerative process, which started at post-spawning and was enhanced during the testicular involution stage, when vitellogenic oocytes appeared in the ovary accompanied by a progressive increase in the ovarian index. However, only 40% of specimens were females in the third RC. Leukocytes (acidophilic granulocytes, macrophages and lymphocytes were present in the gonad and acidophilic granulocyte infiltration occurred during the last two stages. At

  19. 脑缺血对新生大鼠脑室下区神经新生的影响%Neurogenesis in the SVZ of the neonatal rats with brain ischemia injury

    Institute of Scientific and Technical Information of China (English)

    崔曙东; 周文浩; 孙金峤; 陈超; 曹云; 杨毅; 邵肖梅

    2008-01-01

    Objective To investigate the differentiation of neural stem cells in the subventrieular zone(SVZ)of the 3-day-old neonatal rats with ischemic brain injury and to provide the experimental evidences on endogenous repair mechanism of premature brain injury after ischemia. Methods Thirty-two 3-day-old rats were divided into experimental group and the control group.Rats in the experimental group were subjected to bilateral common arteries occlusion.while those in control group were not.All rats were administrated 5-bromodeoxyuridine(BrdU)50 mg/kg by intraperitoneal injection twice daily during 5 to 7 days after the operation.At 14 days and 28 days after operation,rats were sacrificed and their brains were collected.The changes of newborn cells in the SVZ of brain were observed by marking the BrdU,neuronal class Ⅲ β-tubulin(TuJ1),oligodendrocyte O antigen-4 (O4)and glial fibrillary acidic protein(GFAP)with immunofluorescence. Results Compared with the control group,the number(7800±800 vs 4200±700,10 700±1400 vs 4600±600)of newborn neuron(BrdU+/TuJl+)and the number(6100±1000 vs 2600±500,7300±1400 vs 2800±800)of newborn oligodendrocytes(BrdU+/O4+)per field increased significantly at 14 days and 28 days after operation in the experimental group,so did the number of newborn astrocytes per field(4500±700 vs 2700±500,6700±1100 vs 3000±600)respectively(P<0.01). Conclusions The number of neurons,oligodendroeytes and atrocytes in the SVZ increased after brain ischemia,which suggested that SVZ might have the function of repair after brain ischemia injury.%目的 观察3日龄新生大鼠缺血性脑损伤后脑室下区(subventricular zone,SVZ)新生细胞的变化,探讨未成熟脑缺血性损伤后的内源性修复机制. 方法 3日龄新生SD大鼠32只随机分为实验组和对照组,每组16只.实验组结扎双侧颈总动脉,对照组不结扎.两组大鼠均于术后5~7 d给予5-溴脱氧尿嘧啶(5-bromodeoxyuridine,BrdU),每次50 mg

  20. The role of the adventitia in the arterial response to angioplasty: the effect of intravascular radiation

    International Nuclear Information System (INIS)

    Purpose: In the current series of experiments we have characterized cell proliferation leading to vascular lesion formation in a porcine model for post-angioplasty restenosis and examined the mechanism of action of intravascular beta irradiation in the prevention of lesion formation in this model. Methods and Materials: Juvenile male pigs were subjected to balloon overstretch injury of the left anterior descending and circumflex coronary arteries using clinical angioplasty catheters. Proliferating cells were labelled by injections of 50 mg/kg of bromo-deoxyuridine (BrDU) 24, 16 and 8 hrs prior to sacrifice and were detected by immunohistochemistry using a specific antibody to BrDU. In some cases, BrDU was given as a pulse 3 days after angioplasty and the animals sacrificed on day 14 to follow the migration of the cells which had proliferated earlier. Characterization of the proliferating cells was performed by immunohistochemistry using antibodies to specific cytoskeletal proteins specific for smooth muscle cells and myofibroblasts. Some vessels were treated at the time of angioplasty with 14 or 28 Gy (to a depth of 2 mm) intravascular irradiation using a flexible catheter with a pure beta emitter 90 SR/Y and the effect on cell proliferation and terminal transferase-mediated UTP nick-end labelling (TUNEL) examined 3 or 7 days later. Results: The first major site of cell proliferation between 2-3 days after angioplasty is the adventitia and not the medial wall. Seven days after angioplasty cell proliferation is predominant in the neointima and is reduced in the media and adventitia. Differential staining with antibodies directed against smooth muscle alpha actin and other cytoskeletal proteins indicates that the proliferating adventitial cells are myofibroblasts. Pulse label studies with BrDU indicates that the proliferating adventitial myofibroblasts migrate into the neointima and contribute to the mass of the restenosis lesion. Fourteen days after angioplasty the

  1. Pharmacological studies of the mechanism and function of interleukin-1β-induced miRNA-146a expression in primary human airway smooth muscle

    Directory of Open Access Journals (Sweden)

    Jiang Xiaoying

    2010-06-01

    Full Text Available Abstract Background Despite the widespread induction of miR-146a during the innate immune response little is known regarding its biogenesis, function and mechanism. We have therefore examined the role of miR-146a during the interleukin (IL-1β-stimulated IL-6 and IL-8 release and proliferation in primary human airway smooth muscle (HASM cells. Methods HASM cells were isolated from human lung re-section, cultured to a maximum of 3 - 6 passages and then exposed to IL-1β. miR-146a expression were determined by qRT-PCR, IL-6 and IL-8 release by ELISA and proliferation using bromodeoxyuridine incorporation. The role of NF-κB and the MAP kinase pathways was assessed using pharmacological inhibitors of IKK2 (TPCA-1, JNK (SP600125, p38 MAP kinase (SB203580 and MEK-1/2 (PD98059. miR-146a function was determined following transfection of HASM with inhibitors and mimics using Amaxa electroporation. Results IL-1β induced a time-dependent and prolonged 100-fold induction in miR-146a expression, which correlated with release of IL-6 and IL-8. Exposure to IL-1β had no effect upon HASM proliferation. Pharmacological studies showed that expression of primary miR-146a was regulated at the transcriptional levels by NF-κB whilst post-transcriptional processing to mature miR-146a was regulated by MEK-1/2 and JNK-1/2. Functional studies indicated that IL-1β-induced miR-146a expression does not negatively regulate IL-6 and IL-8 release or basal proliferation. However, inhibition of IL-1β-induced IL-6 and IL-8 release was observed at the super-maximal intracellular miR-146a levels obtained by transfection with miR-146a mimics and indicates that studies using miRNA mimics can produce false positive results. Mechanistic studies showed that in the presence of super-maximal levels, the action of miR-146a mimics was mediated at a step following IL-6 and IL-8 mRNA transcription and not through down-regulation of IL-1 receptor associated kinase 1 (IRAK-1 and TNF

  2. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells and Schwann cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Gang; HU Yan-rong; WAN Hong; XIA Lei; LI Jun-hua; YANG Fei; QU Xue; WANG Shen-guo; WANG Zhong-cheng

    2010-01-01

    Background The most important objective of transplant studies in the injured spinal cord has been to provide a favorable environment for axonal growth. Moreover, the continuing discovery of new grafts is providing new potentially interesting transplant candidates. Our purpose was to observe the morphological and functional repair effects of the co-transplantation of neural stem cell (NSC), Schwann ceils (SCs) and poly lactide-co-glycolide acid (PLGA) on the spinal cord injury of rats.Methods A scaffold of PLGA was fabricated. NSCs and SCs were cultured, with the NSCs labeled with 5-bromodeoxyuridine, and the complex of NSC/PLGA or NSC+SCs/PLGA were constructed. Thirty-six Wistar rats were randomly divided into three groups: group A (transplantation of PLGA), group B (transplantation of NSC/PLGA) and group C (transplantation of NSC+SCs/PLGA). The 3 mm length of the right hemicord was removed under the microscope in all rats. The PLGA or the complex of PLGA-celIs were implanted into the injury site. Basso-Beattie-Bresnahan (BBB)locomotion scores, motor and somatosensory evoked potential of lower limbs were examined to learn the rehabilitation of sensory and motor function at 4 weeks, 8 weeks, 12 weeks and 24 weeks after injury. All the recovered spinal cord injury (SCI) tissues were observed with HE staining, immunohistochemistry, and transelectronmicroscopy to identify the survival, migration and differentiation of the transplanted cells and the regeneration of neural fibres at 4 weeks, 8 weeks,12 weeks and 24 weeks after injury.Results (1) From 4 weeks to 24 weeks after injury, the BBB locomotion scores of cell-transplanted groups were better than those of the non-cell-transplanted group, especially group C (P <0.05). The amplitudes of the somatosensory evoked potential (SEP) and motor-evoked potential (MEP) were improved after injury in groups B and C, but the amplitude of SEP and MEP at 4 weeks was lower than that at 12 weeks and 24 weeks after injury. Compared

  3. Differentiation of human dermal-derived multipotent stem cells into epidermal cells in diabetic wound%糖尿病模型皮肤创面中人真皮多能干细胞向表皮细胞的分化

    Institute of Scientific and Technical Information of China (English)

    高新宇; 赵李平; 柯昌能; 王明刚; 钟晓红

    2011-01-01

    BACKGROUND: Differentiation of stem cells is closely related to the microenvironment. Therefore, it is assumed that human dermal-derived multipotent stem cells may have a potential to differentiate into epidermal cells in skin wound.OBJECTIVE: To explore the feasibility of human dermal-derived multipotent stem cells differentiating into epidermal cells under the microenvironment of diabetic wound.METHODS: Human dermal-derived multipotent stem cells were isolated and cultured. Diabetic naked mouse models were prepared. The third to fifth generation of human dermal-derived multipotent stem cells labeled with 5-bromodeoxyuridine (5-BrdU)were injected into the skin tissue surrounding the wound of diabetic mouse models. The specimens were harvested from the wound tissues for paraffin embedding, and serial sections were made at the 2nd and 3rd week after transplantation for BrdU and keratin staining.RESULTS AND CONCLUSION : Not only BrdU positive cells aggregated in the epidermis but also some positive cells expressed keratin simultaneously in the epidermis. During the cou rse of diabetic wound healing, human dermal-derived multipotent stem cells have the potential to differentiate into epidermal cells.%背景:研究表明,干细胞分化为何种细胞与其所处的微环境密切相关.因此设想:人真皮多能干细胞处于皮肤创面的微环境中,可能具有向人皮肤细胞转化的潜能.目的:探讨糖尿病皮肤创面微环境中的人真皮多能干细胞分化为表皮细胞的可能性.方法:分离培养人真皮多能干细胞并制作糖尿病裸鼠模型皮肤创面,将标记5-BrdU的第3~5代人真皮多能干细胞以注射方式回植入糖尿病裸鼠创面组织周围,分别于注射后2,3周取材,常规石蜡包埋、连续切片,行BrdU和角蛋白免疫组织化学染色.结果与结论:BrdU阳性细胞出现在表皮中,且连续切片中部分BrdU阳性细胞也同时表达角蛋白.说明在糖尿病创面愈合过程中,人真皮

  4. Relations of proliferative activities of gastric carcinoma cells to lymphatic involvement, venous invasion and prognosis

    Institute of Scientific and Technical Information of China (English)

    吴云飞; 徐惠绵; 陈峻青

    2004-01-01

    Background This study was to evaluate bivariate bromodeoxyuridine(BrdUrd)/DNA flow cytometric analysis in detection of gastric carcinoma and to study the relations of cellular BrdUrd labeling indices (LI), G2/M-phase fraction(G2/MPF) and DNA ploidy pattern to lymphatic involvement, venous invasion and prognosis.Methods Fresh tumor samples from 60 patients with gastric carcinoma were analyzed by bivariate BrdUrd/DNA flow cytometry. The results were correlated with lymphatic vessel invasion, lymphatic node metastasis, the number of matastatic lymphatic nodes, and venous invasion. Propidium iodide (PI) was used as a fluorescent probe for total cellular DNA, and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescent-labeled goat anti-mouse antibody was used as a second antibody. S-phase fractions were measured by in vitro BrdUrd labeling, and DNA ploidy and G2/MPF were also measured. Comparison of survival was performed with the log-rank test, the Chi-square test for qualitative data, and Student's t test for quantu data. Results BrdUrd LI and G2/MPF values were significantly higher in tumors with lymphatic vessel invasion than in those without invasion respectively (P<0.01); the patients who had tumors with lymphatic vessel invasion showed a significantly poor prognosis (P<0.01). Both BrdUrd LI and G2/MPF values were significantly higher in tumors with lymphatic node metastasis than in those without metastasis (P<0.01). A statistical significant difference was noted in the 5-year survival rates between the patients with lymph node metastasis and those without metastasis. Compared with diploid carcinoma, the incidence of lymph node metastasis was significantly higher in aneuploid carcinoma (P<0.05), and the patients with aneuploid carcinoma showed a significantly poor prognosis (P<0.05). BrdUrd LI was significantly higher in patients with more than 5 metastatic lymph nodes than those with 1-4 metastatic lymph nodes (P<0

  5. Cytotoxicity of dental composite (co)monomers and the amalgam component Hg{sup 2+} in human gingival fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Reichl, Franz-Xaver; Simon, Sabine; Esters, Magalie; Seiss, Mario [Ludwig-Maximilians-University of Munich, Walther-Straub-Institute of Pharmacology and Toxicology, Munich (Germany); Kehe, Kai [Bundeswehr Institute of Pharmacology and Toxicology, Munich (Germany); Kleinsasser, Norbert [University of Regensburg, Department of Otolaryngology - Head and Neck Surgery, Regensburg (Germany); Hickel, Reinhard [Ludwig-Maximilians-University, Department of Operative Dentistry and Periodontology, Munich (Germany)

    2006-08-15

    Unpolymerized resin (co)monomers or mercury (Hg) can be released from restorative dental materials (e.g. composites and amalgam). They can diffuse into the tooth pulp or the gingiva. They can also reach the gingiva and organs by the circulating blood after the uptake from swallowed saliva. The cytotoxicity of dental composite components hydroxyethylmethacrylate (HEMA), triethyleneglycoldimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and bisglycidylmethacrylate (Bis-GMA) as well as the amalgam component Hg{sup 2+} (as HgCl{sub 2}) and methyl mercury chloride (MeHgCl) was investigated on human gingival fibroblasts (HGFs) at two time intervals. To test the cytotoxicity of substances, the bromodeoxyuridine (BrdU) assay and the lactate dehydrogenase (LDH) assay were used. The test substances were added in various concentrations and cells were incubated for 24 or 48 h. The EC{sub 50} values were obtained as half-maximum-effect concentrations from fitted curves. Following EC{sub 50} values were found [BrdU: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 8.860 (0.440)/6.600(0.630), TEGDMA 1.810(0.130)/1.220(0.130), UDMA 0.120(0.010)/0.140(0.010), BisGMA 0.060(0.004)/0.040(0.002), HgCl{sub 2} 0.015(0.001)/0.050(0.006), and MeHgCl 0.004(0.001)/0.005(0.001). Following EC{sub 50} values were found [LDH: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 9.490(0.300)/7.890(1.230), TEGDMA 2.300(0.470)/1.950(0.310), UDMA 0.200(0.007)/0.100(0.007), BisGMA 0.070(0.005)/0.100(0.002), and MeHgCl 0.014(0.006)/0.010(0.003). In both assays, the following range of increased toxicity was found for composite components (24 and 48 h): HEMA < TEGDMA < UDMA < BisGMA. In both assays, MeHgCl was the most toxic substance. In the BrdU assay, Hg{sup 2+} was about fourfold less toxic than MeHgCl but Hg{sup 2+} was about fourfold more toxic than BisGMA. In the BrdU test, a significantly (P<0.05) decreased toxicity was observed for Hg{sup 2+} at 48 h, compared to the 24 h

  6. Histopathologic characterization of the BTBR mouse model of autistic-like behavior reveals selective changes in neurodevelopmental proteins and adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Stephenson Diane T

    2011-05-01

    Full Text Available Abstract Background The inbred mouse strain BTBR T+ tf/J (BTBR exhibits behavioral deficits that mimic the core deficits of autism. Neuroanatomically, the BTBR strain is also characterized by a complete absence of the corpus callosum. The goal of this study was to identify novel molecular and cellular changes in the BTBR mouse, focusing on neuronal, synaptic, glial and plasticity markers in the limbic system as a model for identifying putative molecular and cellular substrates associated with autistic behaviors. Methods Forebrains of 8 to 10-week-old male BTBR and age-matched C57Bl/6J control mice were evaluated by immunohistochemistry using free-floating and paraffin embedded sections. Twenty antibodies directed against antigens specific to neurons, synapses and glia were used. Nissl, Timm and acetylcholinesterase (AchE stains were performed to assess cytoarchitecture, mossy fibers and cholinergic fiber density, respectively. In the hippocampus, quantitative stereological estimates for the mitotic marker bromodeoxyuridine (BrdU were performed to determine hippocampal progenitor proliferation, survival and differentiation, and brain-derived neurotrophic factor (BDNF mRNA was quantified by in situ hybridization. Quantitative image analysis was performed for NG2, doublecortin (DCX, NeuroD, GAD67 and Poly-Sialic Acid Neural Cell Adhesion Molecule (PSA-NCAM. Results In midline structures including the region of the absent corpus callosum of BTBR mice, the myelin markers 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase and myelin basic protein (MBP were reduced, and the oligodendrocyte precursor NG2 was increased. MBP and CNPase were expressed in small ectopic white matter bundles within the cingulate cortex. Microglia and astrocytes showed no evidence of gliosis, yet orientations of glial fibers were altered in specific white-matter areas. In the hippocampus, evidence of reduced neurogenesis included significant reductions in the number of

  7. Effect of treadmill exercise on neurogenesis in the hippocampus of acute epileptic rats%运动对急性癫痫大鼠海马神经发生的影响

    Institute of Scientific and Technical Information of China (English)

    温晓妮; 李红; 李萍; 尤勇

    2012-01-01

    Objective To explore the effect of moderate exercise on neurogenesis in the hippocampus of acute epileptic rats. Methods The single-label immunofluorescence with confocal microscope was used to observe the expression of bromodeoxyuridine (BrdU) in the hippocampus of acute epileptic rats after moderate treadmill exercise for 30 min once a day for 7 consecutive days. Semi-quantitative analysis of the expression of nerve growth factor (NGF) gene in the hippocampus of acute epileptic rats was detected by RT-PCR. The correlation between NGF gene expression and BrdU positive cells in the hippocampus was explored. Results The expression of BrdU positive cells in the dentate gyrus of epileptic rats with exercise decreased significantly compared with those without exercise (P<0.01). The expression of hippocampal NGF gene of epileptic rats with exercise was down-regulated compared with those without exercise (P<0.01). The expression pattern of NGF gene in the hippocampus and expression of BrdU positive cells in the dentate gyrus was the same. Conclusion Moderate treadmill exercise can decrease the expression of newborn neuron BrdU in the dentate gyrus of acute epileptic rats. Hippocampal NGF expression may improve the hippocampal neurogenesis of epileptic rats.%目的 探讨中等负荷运动对急性癫痫大鼠海马神经发生的影响.方法 应用免疫荧光单标记和激光共聚焦显微镜观察癫痫大鼠在中等负荷运动后海马齿状回新生神经元的5-溴脱氧尿核苷(BrdU)表达情况;应用RT-PCR半定量分析癫痫大鼠在中等负荷运动后海马神经生长因子(neurotrophic growth factor,NGF)的表达情况.结果 运动组癫痫大鼠海马齿状回BrdU的表达比未运动组癫痫大鼠表达减少,差异有统计学意义(P<0.01);运动组癫痫大鼠海马NGF基因表达比未运动组癫痫大鼠也下调,差异有统计学意义(P<0.01),且海马NGF基因表达与齿状回BrdU的表达变化相一致.结论 中等负荷的

  8. Predisposition to cancer and radiosensitivity

    Directory of Open Access Journals (Sweden)

    P. Pichierri

    2000-12-01

    Full Text Available Many cancer-prone diseases have been shown to be radiosensitive. The radiosensitivity has been attributed to pitfalls in the mechanisms of repair of induced DNA lesions or to an impaired cell cycle checkpoint response. Although discrepancies exist in the results obtained by various authors on the radiosensitivity of individuals affected by the same disease, these can be attributed to the large variability observed already in the response to radiation of normal individuals. To date three test are commonly used to assess radiosensitivity in human cells: survival, micronucleous and G2 chromosomal assay. The three tests may be performed using either fibroblasts or peripheral blood lymphocytes and all the three tests share large interindividual variability. In this regard a new approach to the G2 chromosomal assay which takes into account the eventual differences in cell cycle progression among individuals has been developed. This new approach is based on the analysis of G2 homogeneous cell populations. Cells irradiated are immediately challenged with medium containing bromodeoxyuridine (BrdUrd. Then cells are sampled at different post-irradiation times and BrdUrd incorporation detected on metaphases spread and the scoring is done only at time points showing similar incidence of labelled cells among the different donors. Using this approach it has been possible to reduce the interindividual variability of the G2 chromosomal assay.Muitas doenças que predispõem ao câncer têm se mostrado radiossensíveis. A radiossensibilidade tem sido atribuída a problemas nos mecanismos de reparo de lesões de DNA induzidas ou a uma resposta alterada no "checkpoint" do ciclo celular. Embora existam discrepâncias entre os resultados obtidos por vários autores quanto à radiossensibilidade de indivíduos afetados pela mesma doença, essas discrepâncias podem ser atribuídas à grande variabilidade observada já na resposta de indivíduos normais à radia

  9. 人胚胎纹状体来源神经干细胞的体外培养**★%In vitro culture of human embryonic striatum-derived neural stem cells**★

    Institute of Scientific and Technical Information of China (English)

    樊明超; 王巧玲; 刘克; 张欣; 关云谦; 孙鹏

    2013-01-01

    BACKGROUND: Neural stem cells are always derived from animals, and unsuitable for human transplantation treatment. OBJECTIVE: To explore the in vitro culture methods of human embryonic striatum-derived neural stem cells, and to observe the biological characteristics. METHODS: The human embryonic striatum were separated from the embryo at a gestational age of 8-16 weeks that received induction of labor with water bag, and then the embryonic striatum was in vitro cultured in the serum-free Dulbecco’s modified Eagle’s medium. The cells were passaged after neurospheres formation, and then the cells were induced to differentiation with the Dulbecco’s modified Eagle’s medium/F12 containing 10% fetal bovine serum. RESULTS AND CONCLUSION: The in vitro cultured human embryonic striatum-derived neural stem cells grew rapidly and could express nestin. Colony formation assay showed the cel clone formation rate was 6.0%-7.0%. 5-Bromodeoxyuridine incorporation assay showed the cel proliferation rate was 37.9%. Immunofluorescence staining showed that the cells after induction and differentiation could express Tuj-1, glial fibril ary acidic protein and nestin, but not express myelin basic protein. The results indicate that human embryonic striatum-derived neural stem cells cultured in the serum-free medium can maintain their biological characteristics and have self-renewal capacity, and the cells can differentiate into the neurons and astrocytes induced by the fetal bovine serum.%  背景:目前神经干细胞多由动物获得,不适合人类临床移植治疗。目的:探索体外环境下人胚胎纹状体来源神经干细胞的培养方法,同时观察其生物学特性。方法:取经水囊引产的孕8-16周人胚胎纹状体,体外用无血清 DMEM 培养基进行培养,待细胞形成神经球后进行传代,并应用含体积分数10%胎牛血清的 DMEM/F12培养液进行诱导分化。结果与结论:体外培养的人胚胎纹状体来

  10. Effects of mitomycin C on infiltration of polymorphonuclear leukocytes after epithelial scrape injury in the mouse cornea Efeito da mitomicina C na infiltração de leucócitos polimorfonucleares após lesão epitelial em córnea de camundongo

    Directory of Open Access Journals (Sweden)

    Ana Cecília Souza Leão Escarião

    2008-12-01

    Full Text Available PURPOSE: To determine whether mitomycin C (MMC alters appearance and disappearance of polymorphonuclear leucocytes (PMN in the cornea stroma, using an epithelial scrape injury in eye mouse model. METHODS: Twenty-mice underwent mechanical epithelium debridement in the central cornea using 20% ethanol. After the scrape, the right eye received 0.02% MMC for one minute, while the left eye received physiological saline. The animals were sacrificed on days 1, 2, 5, and 14 after surgery, and corneal whole mounts were prepared for histology. PMN distribution was analyzed in digitized microscope images. Cell division in the cornea was determined by immunohistochemical detection of bromodeoxyuridine (BrdU, which was injected intraperitoneally before the mice were sacrificed. RESULTS: Epithelial scrape injury triggered infiltration of PMNs into the corneal stroma. An analysis of PMN distribution revealed that there was no difference between eyes treated with and without MMC at all time points. BrdU labeling showed that 0.02% MMC for one minute blocked keratocyte proliferation completely. CONCLUSION: MMC treatment regimen, which is common in clinical practice, inhibits keratocyte proliferation during wound healing, but when used at 0.02% for one minute, it does not affect PMN infiltration into the corneal stroma, and subsequent movement toward the injury site, or the disappearance of PMNs from the stroma, in the mouse epithelial injury model.OBJETIVO: O objetivo do estudo foi determinar se a mitomicina C (MMC altera o aparecimento dos leucócitos polimorfonucleares (PMN no estroma corneano após abrasão epitelial central, utilizando olhos de camundongo como modelo. MÉTODOS: Vinte camundongos foram submetidos à abrasão epitelial em córnea central utilizando etanol a 20%. Após a lesão, o olho direito recebeu MMC a 0,02% por 1 minuto, enquanto o olho esquerdo recebeu solução salina. Os animais foram sacrificados em 1, 2, 5 e 14 dias após a cirurgia e

  11. Use of a temperature-sensitive p53 mutant to evaluate mechanisms of 5-fluorodeoxyuridine-mediated radiosensitization

    International Nuclear Information System (INIS)

    Purpose/Objective: Evidence exists that fluorodeoxyuridine (FdUrd)-mediated radiosensitization occurs in HT29 human colon carcinoma cells (which are p53 mutant) when these cells progress past the G1/S boundary in the presence of the drug. It has been demonstrated that wild type p53 levels increase following fluoropyrimidine treatment and that G1 arrest is associated with increased p53 levels. We hypothesized that the restoration of wild type p53 function might restore G1/S arrest after FdUrd treatment, and that this would prevent FdUrd-mediated radiosensitization. Similarly, we hypothesized that cells containing wild type p53 would not be radiosensitized by FdUrd. Materials and Methods: Two clones of HT29 human colon cancer cells (ts29-A and ts29-G) containing murine temperature-sensitive p53 were constructed using electroporation and Geneticin selection. Incubation of these cells at the permissive temperature of 32 deg. C produces wild type p53 function and at the non permissive temperature of 38 deg. C causes mutant p53 function. A G418 resistant control cell line was also constructed (HT29neo). Cells were incubated at either 32 deg. C or 38 deg. C for 24 hours prior to irradiation and with FdUrd (100 nM) or medium only during the last 14 hours of the temperature shift. To assess progression into S phase, single-parameter (propidium iodide (PI)) and two-parameter (PI and bromodeoxyuridine) flow cytometry were performed at the end of drug exposure. A standard clonogenic assay was used. Results: We found that when ts29-A and ts29-G cells were incubated at the non-permissive (inactive p53 conformation) temperature, they progressed into S phase following exposure to FdUrd and were radiosensitized (enhancement ratio 1.5) to a degree similar to that seen in parental HT29 cells. Cells incubated at the permissive (wild-type p53 conformation) temperature demonstrated G1 arrest, S phase depletion, and G2 arrest. In addition, FdUrd-mediated radiosensitization was abrogated

  12. 小鼠脑组织发育期细胞增生与分化的改变%Changes of cell proliferation and differentiation in the developing brain of mouse

    Institute of Scientific and Technical Information of China (English)

    邱林; 朱长连; 王小阳; 徐发林

    2007-01-01

    Objective To investigate the cell proliferation and differentiation in the developing brain of mouse. Methods C57/BL6 mice were divided into 3 groups at random. Bromodeoxyuridine (BrdU) was injected into the brains in different development periods once a day for 7 d. The brains were retrieved 4 weeks after the last BrdU injection. Immunohistochemical and immunofluorescent studies were carried out for detecting cell proliferation (BrdU) and cell differentiation (NeuN, APC, Iba1, and S100β), respectively. Results The number of BrdU labeled cells decreased significantly with the development of the brain. Cell proliferation was prominent in the cortex and striatum. A small portion of BrdU and NeuN double labeled cells could be detected in the cortex at the early stage of development, and in the striatum and CA of the hippocampus in all groups. The majority of BrdU labeled cells were neuroglia, and the number of neuroglia cells decreased dramatically with brain maturation. Neurogenesis is the major cytogenesis in the dentate gyrus. Conclusion These results demonstrated that cell proliferation, differentiation and survival were age and brain region related.%目的 探讨小鼠脑组织发育期间的细胞增生与分化.方法 C57/BL6小鼠分别于出生后10大(P10)、17天(P17)、24天(P24)不同脑发育期,每天注射新生细胞标记物5-溴脱氧尿嘧啶核苷(BrdU),连续注射7天,并分别于末次注射后四周将小鼠处死、取脑.采用免疫组化染色及免疫荧光染色分别检测细胞增生(BrdU)与细胞分化(NeuN、APC、Iba1和S100β).结果 细胞增生随着脑组织发育快速下降,并以皮层和纹状体区细胞增生最显著.皮层在发育早期以及纹状体和海马CA区在发育各期检测到极少数为新生神经元细胞,多数分化为胶质细胞;海马齿状回以神经元细胞再生为主;胶质细胞的再生随脑组织发育的成熟而逐渐减少.结论 研究证实小鼠脑组织细胞的增生、分化以

  13. Ultrastructure observation of rhesus bone marrow mesenchymal stem cell after transplantation of cornea%恒河猴骨髓间充质干细胞角膜移植后的超微结构观察

    Institute of Scientific and Technical Information of China (English)

    韦春玲; 孙晓梅; 杨忠昆; 代解杰; 刘海; 吉祥; 胡竹林

    2011-01-01

    连接较前紧密,均为单细胞层;角膜植片内表面可见BrdU抗体染色阳性的细胞;对照组扫描电子显微镜检测角膜植片内皮表面无细胞生长,弹性纤维裸露,BrdU染色阴性。 结论 BMSCs通过离心沉淀法移植于角膜内表面可以存活并增生为单细胞层。%Background The quest to look for seed cells is a hot spot of cornea transplant research in solving the problem of the lack of donor. Bone marrow mesenchymal stem cells(BMSCs) have been successfully induced into retinal ganglion cells(RGCs) in vivo,but the successful induction of BMSCs into corneal endothelial cells has not been reported.Objective This experiment was to study the transplantation of BMSCs on corneal endothelial surface using the splitting Descemet's membrane. Methods Four healthy adult rhesus monkeys were divided into the experimental group ( 3 monkeys) and control group ( 1 monkey). Mesenchymal stem cells (MSCs) were isolated from bone marrow by density gradient centrifugation combined with adhering means. The cultured cells were identified by flow cytometry and its ability to differentiate was determined by allowing them to differentiate into adipocytes in vitro and labeled by 5-bromodeoxyuridine ( BrdU ) for subsequent identification. Corneal grafts of 7 mm in size with tearing of the Descemet' s membrane were prepared in the experimental group and control group. After labeling by 5-bromodeoxyuridine( BrdU ) ,cultured cells were transplanted onto the endothelial surface of cornea grafts in the experimental group, but no cultured cells were seeded in the graft of the control group. The corneal grafts were then sutured in situ, and were removed 1,2 or 3 months after operation to examine the distribution and connection between transplanted cells and their morphologic changes under the electron microscope. Results High purity MSCs were harvested by density gradient centrifugation combined with adhering method. Cultured cells reached confluency

  14. Heterotrophic Soil Respiration in Warming Experiments: Using Microbial Indicators to Partition Contributions from Labile and Recalcitrant Soil Organic Carbon. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Bradford, M A; Melillo, J M; Reynolds, J F; Treseder, K K; Wallenstein, M D

    2010-06-10

    The central objective of the proposed work was to develop a genomic approach (nucleic acid-based) that elucidates the mechanistic basis for the observed impacts of experimental soil warming on forest soil respiration. The need to understand the mechanistic basis arises from the importance of such information for developing effective adaptation strategies for dealing with projected climate change. Specifically, robust predictions of future climate will permit the tailoring of the most effective adaptation efforts. And one of the greatest uncertainties in current global climate models is whether there will be a net loss of carbon from soils to the atmosphere as climate warms. Given that soils contain approximately 2.5 times as much carbon as the atmosphere, a net loss could lead to runaway climate warming. Indeed, most ecosystem models predict that climate warming will stimulate microbial decomposition of soil carbon, producing such a positive feedback to rising global temperatures. Yet the IPCC highlights the uncertainty regarding this projected feedback. The uncertainty arises because although warming-experiments document an initial increase in the loss of carbon from soils, the increase in respiration is short-lived, declining to control levels in a few years. This attenuation could result from changes in microbial physiology with temperature. We explored possible microbial responses to warming using experiments and modeling. Our work advances our understanding of how soil microbial communities and their activities are structured, generating insight into how soil carbon might respond to warming. We show the importance of resource partitioning in structuring microbial communities. Specifically, we quantified the relative abundance of fungal taxa that proliferated following the addition of organic substrates to soil. We added glycine, sucrose, cellulose, lignin, or tannin-protein to soils in conjunction with 3-bromo-deoxyuridine (BrdU), a nucleotide analog. Active

  15. rhG-CSF促进体外培养神经干细胞的增殖%rhG-CSF promoting the proliferation of neural stem cells in vitro

    Institute of Scientific and Technical Information of China (English)

    李延兰; 贾德永; 付洁; 刘慧娟

    2011-01-01

    Objective To investigate whether granulocyte colony-stimulating factor ( G-CSF ) has a direct effect on the proliferation of mouse neural stem cells in vitro. Methods Primary mouse neural stem cells( NSCs ) were isolated from Kunming mice and cultured in serum free medium. The third passage NSCs were used in the experiments. NSCs were treated with G-CSF ( 10, 30, 60 , 100 , and 200μg/L ). The proliferation of NSCs was detected by MTT colorimetric assay and bromodeoxyuridine ( BrdU ) incorporation assay. The expressions of STAT3 and p-STAT3 were examined by Western blotting. Results There was the expression of G-CSF receptor on the neural stem cells. G-CSF obviously improved the viabilities of NSCs in a dose-dependent manner. Furthermore, 100μg/L G-CSF yielded the optimal effect. The BrdU-positive cells against total NSCs treated with G-CSF were markedly enhanced. Time course experiments demonstrated that STAT3 phosphorylation by G-CSF was evident after 5 minutes, reaching the maximum after 30 minutes. At 75 minutes, the effect on STAT3 returned to the basal value. Pretreatment with the G-CSF receptor antibody significantly reduced the phosphorylation of STAT3 induced by G-CSF. BrdU incorporation showed that cell proliferation in the presence of G-CSF and neutralizing antibody for G-CSF receptor was similar to that of the reduced growth medium group. Conclusion G-CSF may stimulate the proliferation of NSCs in vitro. Our results provide the cellular basis of the role of G-CSF in NSCs in vitro ,which may serve as a useful reference for future studies of the powerful neuroprotective effect of G-CSF in various types of neurological disorders.%目的 探讨重组人粒细胞集落刺激因子(rhG-CSF)对小鼠胚胎神经干细胞(NSCs)增殖的作用.方法 分离培养小鼠NSCs,通过向培养基中添加G-CSF(10、30、60、100和200μg/L),四甲基偶氮唑蓝(MTT)比色法以及5-溴-2′-脱氧尿苷(BrdU) 免疫荧光染色标记检测NSCs的增殖.免疫印

  16. 骨髓动员单个核细胞向血管内皮细胞和心肌样细胞的转化%Mononuclear cells isolated from mobilized bone marrow differentiate into vascular endothelial cells and cardiomyocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    姚巍; 张秀兰; 姚英; 王卫淑

    2014-01-01

    after treatment, bromodeoxyuridine (BrdU, 50 mg/kg) was successively given to al rats for 4 weeks before they were sacrificed. Myocardial tissues were taken to determine the homing of mononuclear cells and evaluate differentiation of mononuclear cells into cardiomyocytes and vascular endothelial cells using BrdU staining, BrdU/myosin heavy chain double staining, and BrdU/actin double staining. Hematoxylin-eosin staining was used for determination of blood vessel density. RESULTS AND CONCLUSION:G-CSF mobilization increased the number of mononuclear cells that was significantly higher than before treatment (P<0.05). Flow cytometry showed that the number of CD34-positive mononuclear cells in the peripheral blood was higher in the bone marrow mobilization than in the heart failure group (P<0.05). Myocardial CD34 immunofluorescence showed that the heart failure group was negative and the bone marrow mobilization group was positive. In the bone marrow mobilization group, the myocardial tissue was positive for BrdU staining, BrdU/myosin heavy chain double staining and BrdU/actin double staining, while vascular endothelial cells in the region of myocardial injury was positive for BrdU;conversely, the heart failure group was negative. The density of blood vessels in the bone marrow mobilization group was significantly higher than that in the heart failure group (P<0.001). These findings indicate that bone marrow mobilization increases the number of mononuclear cells, and these cells are homing to myocardial injury, thereby playing a repair role in the myocardium and vascular tissue of heart failure rats with congestive cardiomyopathy.

  17. 大鼠骨髓间质干细胞的BrdU体外标记和体内示踪研究%In vitro Analysis and in vivo Tracing of BrdU-labeled Rat Bone Marrow Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    张明鸣; 贾贵清; 程惊秋; 杨平; 陆燕蓉; 伍晓汀

    2012-01-01

    Objectives To investigate the optimal condition of bromodeoxyuridine (BrdU) labeling for bone marrow mesenchymal stem cells (BMSCs) in vitro and the feasibility of in vivo tracing of BrdU-labeling BMSCs. Methods BMSCs were isolated from Wistar rats and were in vitro routinely cultured. The third passage BMSCs was used for identification of special surface antigens by immunohistochemical methods. The purified BMSCs were incubated with BrdU at different concentrations for different incubating time to investigate optimal BrdU concentration and incubating time for cell labeling. The cell labeling index of BrdU was calculated with immunohistochemical analysis. BMSCs labeled BrdU were injected into damaged gastric mucosa of rats by micro injector. The colonization of BMSCs labeled BrdU in gastric mucosa was viewed. Results After purification and proliferation, the primary cultured BMSCs were uniformly long spindle-shapped form and formed cell colony, which showed the characteristics of stem cell. Immunocytochemistry showed BMSCs were positive for CD44 and CD90, while negative for CD14, CD45. The labeling rate of BrdU increased with the labeling time lasting and reached its height at 48 h. After incubating 48 and 72 hours, the labeling rate of BrdU with a concentration of 10 μmol/L was higher than that of BrdU with a concentration of 5 μmol/L (P 0. 05). In addition, the BrdU labeling could be detected after five consecutive passages and the labeling time could keep 21 d. The pathological observation demonstrated that BrdU-labeled BMSCs could colonize the damaged gastric mucosa with normal morphologic characteristics during observation period. Conclusion BrdU labeling might be a feasible method for dynamic observation of the migration, growth and differentiation of migrating BMSCs in colonizing sites.%目的 探讨骨髓间质干细胞(BMSCs)的体外标记和体内示踪技术.方法 分离培养并鉴定大鼠BMSCs,进行5-溴脱氧尿嘧啶核苷(BrdU)标记后,体

  18. Somatic cell banking - An alternative technology for conservation of endangered sheep breeds

    International Nuclear Information System (INIS)

    cells but these were removed mechanically as well as enzymatically to get pure fibroblasts. Sub-culturing or 'splitting cells' was done periodically removing growth media, washing the plate, dissociating the cells and diluting cell suspension in fresh media. Standard growth curve: Whenever, a new batch of culture media supplement was introduced, it was checked for its efficacy for growth of cells in culture and compared with standard growth curve. Goat skin fibroblasts remained in lag phase for initial two days when they settled on the solid surface of culture vessel and then came to log phase when maximum growth took place spanning from the third to the seventh day. As the confluencey level increased and media supplement was depleted, cells stopped dividing and a plateau was attained from the eighth day onwards and then showed decline due to contact inhibition. Cell proliferation index: Under standard culture conditions, skin fibroblast cells divide once in 24 hours but it is rarely achieved in normal culturing. The population doubling time and cell proliferation rate per day were checked at regular interval for quality assessment. For this, ELISA based MTT assay, incorporation of 5-bromo-de-oxyuridine method, and flow cytometer methods were used. Evaluation of cells for ploidy level: During long-term culturing the cells are likely to develop one or other type of chromosomal abnormalities. It must be ensured that the cells in different passages be checked for normal ploidy so that viable clones can be developed from them. Cultures showing increased frequency of aneuploidy or polyploidy must be terminated from further passaging. DNA from cultured somatic cells can be isolated using available DNA isolation kits and checked for its quality on 2% agarose. Cryo-freezing of cells: Cells are best frozen as cell suspension. Healthy culture were always employed to provide the stock to freeze cells. The cells were frozen at controlled freezing rate. The cells were kept at -80

  19. 缺血启动未成熟大鼠脑白质内源性修复功能的研究%Endogenous self-repair in immature white matter induced by ischemia in neonatal rats

    Institute of Scientific and Technical Information of China (English)

    李文娟; 陈惠金; 钱龙华; 毛凤霞

    2012-01-01

    目的 探讨缺血启动未成熟脑白质的内源性修复机制.方法 5日龄Sprague-Dawley新生大鼠随机分为假手术(Sham)组和PVL组.分别于建模后7d及21 d光镜、电镜下评估脑白质病变及髓鞘形成情况,免疫组化检测脑白质O4+少突胶质细胞(OL)前体,观察SVZ区祖细胞的激活、增殖、迁移和分化情况.结果 与Sham组比较,PVL组在建模后7d和21d光镜下脑白质病理均呈轻或重度病变;病理评分均明显增高;髓鞘形成数量明显减少,厚度变薄;免疫组化显示O4+OL前体明显减少.建模48h后,PVL组SVZ区BrdU、NG2共阳性祖细胞明显增殖并向脑室周围迁移,至7d达到高峰;从72 h开始,脑室周围出现呈BrdU、O4共阳性OL前体,至21 d,新生OL 前体明显多于同时段Sham组.结论 缺血可启动新生大鼠脑白质的内源性修复机制,诱导SVZ区胶质源性神经祖细胞激活、增殖、迁移至脑室周围和分化为OL前体.%ischemia in neonatal rats with periventricular leukomalacia ( PVL). Methods Five-day-old neonatal Sprague-Dawley (SD) rats were randomly divided into sham and PVL groups. Rat model of PVL was prepared by ligation of the right common carotid artery following 2 hours of exposure to 8% oxygen. Pathological changes and myelination in the white matter were assessed under light and electron microscopy at 7 and 21 days after PVL. 04-positive OL precursor cells in the white matter were determined with immunofluorescence staining. Activation, proliferation, migration and differentiation of glial progenitor cells in SVZ were observed using immunofluoreacent double labeling of either NG2 ( marker of progenitor cells) and 5-bromodeoxyuridine (BrdU), or 04 (marker of OL precursor cells) and BrdU. Results All rats in the PVL group manifested either mild or severe white matter injury under light microscopy, and had higher pathological scores of white matter compared with the sham group at 7 and 21 days after PVL ( P < 0

  20. 新生期大鼠反复痫性发作的形态学及行为学结果与糖皮质激素水平升高有关%Morphological and behavioral consequences of recurrent seizures in neonatal rats are associated with glucocorticoid levels

    Institute of Scientific and Technical Information of China (English)

    石秀玉; 王纪文; 雷格非; 孙若鹏

    2007-01-01

    Objective It is well documented that epilepsy can increase neurogenesis in certain brain regions and cause behavioral alternations in patients and different epileptic animal models. A series of experimental studies have demonstrated that neurogenesis is regulated by various factors including glucocorticoid (CORT), which can reduce neurogenesis.Most of studies in animal have been focused on adulthood stage, while the effect of recurrent seizures to immature brain in neonatal period has not been well established. This study was designed to investigate how the recurrent seizures occurred in the neonatal period affected the immature brain and how CORT regulated neurogenesis in immature animals.Methods Neonatal rats were subjected to 3 pilocarpine-induced seizures from postnatal day 1 to day 7. Then neurogenesis at different postnatal ages (i.e. P8, P12, P22, P50) was observed. Behavioral performance was tested when the rats were mature (P40), and plasma CORT levels following recurrent seizures were simultaneously monitored. Results Rats with neonatal seizures had a significant reduction in the number of Bromodeoxyuridine (BrdU) labeled cells in the dentate gyrus compared with the control groups when the animals were euthanized on P8 or P12 (P < 0.05); whereas there was no difference between the two groups on P22. Until P50, rats with neonatal seizures had increased number of BrdU-labeled cells compared with the control group (P < 0.05). In Morris water maze task, pilocarpine-treated rats were significantly slower than the control rats at the first and second day, and there were no differences at other days. In probe trial, there was no significant difference in time spent in the goal quadrant between the two groups. Endocrine studies showed a correlation between the number of BrdU positive cells and the CORT level. Sustained increase in circulating CORT levels was observed following neonatal seizures on P8 and P12. Conclusion Neonatal recurrent seizures can

  1. Study on effect of cholecystokinin-A receptor antagonist L-364, 718 in experimental pancreas regeneration model%胆囊收缩素A受体拮抗剂L-364,718在实验性胰腺再生模型中的作用

    Institute of Scientific and Technical Information of China (English)

    劳学军; 曹明溶; 庞钊; 龚瑾

    2005-01-01

    目的研究胆囊收缩素A受体拮抗剂(CCK-RA)在实验性胰腺再生模型中的作用.方法全部24只大鼠,随机分成3组,Wistar大鼠分为假手术Px(-)、手术加使用CCK-RA受体完全拮抗剂Px+CCK-RA和手术但不使用CCK-RA受体完全拮抗剂组Px,5d后测定它们环胆管下端部分湿重、免疫组化观察不同部位的细胞摄取溴脱氧尿苷以及放射免疫方法血清CCK的浓度.结果Px(-)组环胆管下端部分湿重为(86.98±3.51)mg,Px+CCK-RA组为(92.46±6.67)mg,Px组为(210.72±7.39)mg,3组比较差异有显著性(F=772.9,P<0.01);免疫组化观察不同部位的细胞摄取溴脱氧尿苷的永生化指数Px(-)组为1%,Px组为10%,Px+CCK-RA组为5%;Px(-)组中血清CCK的水平为(28.22±2.45)pg/mL,px+CCK-RA组为(53.62±4.49)pg/mL,Px组为(62.85±4.33)pg/mL,3组比较差异有显著性(F=171.8,P<0.01).结论内源性CCK是胰腺再生的基本要素之一,胰腺部分切除可以促进其分泌,这些作用可以被CCK-RA遏制,有关分子机制有待进一步阐明.%[Objective] To study effect of cholecystokinin-A recaptor antagonist on experimental pancreas regeneration model. [Methods] 24 rats were divided into 3 groups, as sham operation Px(-), using CCK-RA antagonist with pancreastectomy Px+CCK-RA, pancreastectomy without using CCK-RA antagonist Px by random method.After 5 days, we detected the samples wet weight of parabiliary segment from the duodenum and cell proliferation index by microscope observation using bromodeoxyuridine (BrdU) labeling,cell proliferation index(LI) and the plasma CCK concentrations were measured. [Results] 24 rats were detected, wet weight of parabiliary segment from the duodenum in Px(-) group is (86.98±3.51) mg, Px+CCK-RA group (92.46±6.67) mg, Px group (210.72±7.39) mg,compared each group have revealed significant difference (F=772.9, P <0.01). In Px(-) group, the LI in acinar cells was approximately 1%, but in Px and Px+CCK-RA group are about 10% and 5%; Plasma CCK