Plant tissue culture regeneration is chiefly regulated by exogenous phytohormones. To stop regeneration and induce undifferentiated callus growth auxins are used. Unfortunately auxins influence many plant responses, most unrelated to development. Using the thymidine analogue 5-bromodeoxyuridine (BrdU) a phytohormone independent means for differentiation inhibition has been developed. Studies were focused on the target site and mechanism of BrdU action. The BrdU inhibited step in development is indicative of a plant response necessary for normal differentiation. BrdU (5-30 uM) interrupts callus growth in all tested plants. Exogenous cytokinin does not restore growth while thymidine and deoxycytidine rescue plant growth and differentiation in the presence of BrdU. Endogenous cytokinin levels are not greatly affected by subtoxic BrdU levels and indicate that cytokinin and BrdU act upon independent sites. Domestic carrot cells were used in further studies. Normally carrot cells are undifferentiated in medium with the auxin 2,4D. When 2,4D is removed, somatic embryogenesis takes place. By including 5 uM BrdU in the hormoneless medium, the cells fail to differentiate. The growth of carrots in 2,4D is not affected by 5 uM BrdU. Thus, BrdU influences growth during differentiation to a greater extent than the growth of callus cells. BrdU is effective in halting development when applied 0-24 hours after differentiation induction. An event required for differentiation (the first and second replications) must take place at this time. BrdU action begins with DNA incorporation. The consequent cellular replication becomes slowed and DNA repair results. At the same time RNA and protein levels are similar in BrdU treated and untreated cultures. BrdU thymidine substitution into DNA increases from 28% (2 days) to 68% (3 days) after embryogenic induction. A second BrdU effect follows DNA incorporation. Factors (MIFs) in the medium of BrdU treated cells arrest differentiation. After BrdU is repaired from the DNA, the cells are only able to differentiate after a medium change. Understanding MIF production could explain why some plants differentiate more readily than others. BrdU provides the means for further study of MIF's in the auxin-free inhibition of development.

We describe a simple, sensitive, nonradioactive, relatively rapid and relatively inexpensive protocol to measure DNA synthesis in cultured cells by a chemiluminescent bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA). We show that it exhibits similar sensitivity and activity as traditional 3H-thymidine incorporation assays and a commercial chemiluminescent BrdU ELISA kit when tested in commonly used cell lines, such as NIH 3T3 cells, mink lung epithelial cells (Mv1LU), and baby hamster kidney (BHK-21) fibroblasts. This assay also exhibits a wider dynamic range than colorimetric BrdU ELISA methods. Besides being a viable, nonradioactive alternative to 3H-thymidine incorporation assays, our BrdU ELISA is less expensive than a commercial chemiluminescent BrdU ELISA kit.

歯科基礎医学会, 高橋茂 = Shigeru Takahashi, YAGレーザー照射によるラット顎下腺の損傷と修復に関する形態学的研究 = Morphological studies on the regeneration of rat submandibular gland following YAG laser irradiation, 歯科基礎医学会雑誌, APR 1993, 35(2), pp.115-146 / Hokkaido University (北海道大学) / 博士 / 歯学

Microbial community interactions within many ecosystems are still relatively unknown. Investigating links between environmental dynamics and shifting pelagic bacterial community structures on the Texas-Louisiana shelf, Eubacterial community profiles of Operational Taxonomic Units (OTUs) were generated using Automated Ribosomal Intergenic Spacer Analysis (ARISA) of the 16S rDNA and 23S rDNA intergenic spacer region. This ITS region is highly variable in both length and sequence. Community diversity was assessed by the comparison of ARISA-generated community fingerprints of samples collected from four distinct regions along the Texas-Louisiana shelf in a cross-shelf pattern on 10m, 20m, and 40m isobaths. Incubations of samples with a thymidine analog, 5-Bromodeoxyuridine (BrdU), allowed for the isolation and analysis of the actively growing subset within the total bacterial population. Community composition was determined through the construction of clone libraries for sequencing and putative phyla affiliation of community 16 rRNA genes. Hydrographic data were also collected for analysis of shifts in microbial community diversity correlated with a variety of influential environmental factors. ARISA profiles of Eubacterial species richness suggest strong distinction between the two communities found within Zones A and C along the Texas-Louisiana Shelf. Further analysis of salinity gradients originating from the two main fluvial sources, the Mississippi and the Atchafalaya Rivers, identified possible sources of variation between the individual communities. Whereas composition of these communities remains discrete between regions, the active subset of the population becomes more similar across the shelf through the summer. Possibly due to undersampling of hypoxic sites, no relationship could be determined between hypoxia formation and the Eubacterial community dynamics. Several OTUs within the communities were identifiable as α - and β - Proteobacteria, Actinobacteria, Synechococcus, Prochlorococcus, and Cytophaga/Flavobacterium/Bacteroides. Through validation studies of 5-Bromodeoxyuridine field sampling, this study indicates the power of BrdU incorporation and ARISA analysis to study a dynamic environmental system and explore the factors that determine the structure of the pelagic community on the Texas-Louisiana Shelf.

Em busca de novas estratégias para o reparo de tecidos pulpares com células-tronco faz-se necessário compreender melhor o nicho em que elas se encontram. Uma forma é observar o mecanismo de ativação sobre estas células durante o reparo de tecidos pulpares após sofrer uma injúria. Assim, foi proposto estudar a localização e composição do nicho das célulastronco da polpa dental através da exposição pulpar induzida in vitro em dentes humanos e in vivo em dentes de ratos, através da incorporação de Bromodeoxiuridina (BrdU) e Iododeoxiuridina (IdU) e co-expressão com proteínas de células-tronco. Para o ensaio in vitro, 33 terceiros molares humanos com rizogênese incompleta foram divididos em 3 grupos: dentes com exposição pulpar induzida e mantidos em cultivo (GCE, grupo cultura exposição) (n=15); dentes sem injúria e mantidos em cultivo (GCC, grupo cultura controle) (n=15); dentes hígidos apenas (GH, grupo hígido) (n=3). Os grupos GCE e GCC, foram cultivados com BrdU (10 l/ml) por 24h e posteriormente analisados após 2, 5 e 14 dias. Para o ensaio in vivo, 12 ratos Wistar receberam IdU (1mg/ml) diluído na água por 30 dias. Após 45 dias foram divididos em 2 grupos: grupo in vivo com exposição pulpar (GIVE) (n=17 dentes) e grupo in vivo controle (GIVC) (n=16 dentes). No GIVE foi realizada a exposição pulpar no primeiro molar e em seguida restaurado com Coltosol. Após 2, 4 e 8 dias pós-operatórios as amostras foram coletadas e processadas para análise histológica e imuno-histoquímica. As proteínas CD90, CD146, PDGFr e BrdU foram analisadas em duas áreas: lesão e centro em molares humanos enquanto, as proteínas CD90, NG2 e IdU foram analisadas em uma área única, em molares de rato. As análises estatísticas foram efetuadas pelos testes de Mann- Whitney e Kruskal-Wallis, p 5%. Em dentes humanos, na área de lesão do grupo GCE, células BrdU positivas foram encontradas nos três períodos, sendo presentes em maior número células PDGFr positivas nos dias 2 e 5 e células CD90 positivas no 14º dia; no centro pulpar, no 2º dia pós injúria houve um número semelhante de células CD90, CD146 e PDGFr positivas, sendo encontradas em maior porcentagem no 5º dia células PDGFr e CD90 positivas e 14º dia células CD90 positivas. No grupo GCC na área equivalente à lesão, células CD90 (2 e 5 dias) e PDGFr (14 dias) positivas foram encontradas em maior percentual; no centro pulpar, o número de células positivas para CD90, CD146 e PDGFr foi semelhante em todos os períodos, sendo encontradas células BrdU positivas no 2º dia. Em molares de ratos, no GIVE houveram mais células NG2 positivas no 2º dia, IdU no 4º dia e CD90 no 8º dia. No GIVC, o CD90 foi altamente expresso em todos os períodos analisados, a proteína NG2 também foi expressa em odontoblastos e CD90 na camada subodontoblástica em molares de rato. Todas as proteínas mencionadas coexistem no tecido pulpar humano e animal formando populações celulares heterogêneas perivasculares e perineurais encontradas em maior concentração em regiões próximas à área de injúria, sugerindo que nichos perivasculares e perineurais participam em conjunto na homeostase pulpar em resposta à injúria. / In pursuit of new strategies for pulp tissue repair with stem cells, it is necessary to better understand the niche in which they are found. One way is to observe the mechanism of these cells activation during the repair of pulp tissues after suffering an injury. Thus, it was proposed to study the location and composition of the dental pulp stem cell niche through induced dental pulp exposure in vitro in human teeth and in vivo in rat teeth by incorporating Bromodeoxyuridine (BrdU) and Iododeoxyuridine (IdU) and co-expression with stem cell proteins. For the in vitro assay, 33 human third molars with incomplete rhizogenesis were divided into 3 groups: teeth with induced pulp exposure and maintained in culture (GCE, group cultured with pulp exposure) (n = 15); healthy teeth maintained in culture (GCC, control cultured group) (n = 15); healthy teeth only (GH, healthy group) (n = 3). GCE and GCC groups were cultured with BrdU (10 l / ml) for 24 h and subsequently analyzed after 2, 5 and 14 days. For the in vivo assay, 12 Wistar rats received IdU (1mg / ml) diluted in water for 30 days. After 45 days were divided into 2 groups: in vivo group with pulp exposure (GIVE) (n = 17 teeth) and in vivo control group (GIVC) (n = 16 teeth). In GIVE, the pulp exposure was performed on the first molar and then restored with Coltosol. After 2, 4 and 8 postoperative days the samples were collected and processed for histological and immunohistochemical analysis. CD90, CD146, PDGFr and BrdU proteins were analyzed in two areas: injury and center in human molars whereas, CD90, NG2 and IdU were analysed in a single area in rat molars. Statistical analyzes were performed by the Mann-Whitney and Kruskal-Wallis tests, p 5%. In human teeth, in the lesion area of the GCE group, BrdU positive cells were found in the three periods, while a greater number of PDGFr positive cells on days 2 and 5 and CD90 positive cells on the 14th day was found; in the pulp center, on the second day after injury there were a similar number of CD90, CD146 and PDGFr positive cells, and found in greater percentage in the 5th day PDGFr and CD90 positive cells and in 14th day CD90 positive cells. In the GCC group, in the area equivalent to injury site, CD90 (2 and 5 days) and PDGFr (14 days) positive cells were found in a higher percentage; in the pulp center, the number of cells positive for CD90, CD146 and PDGFr was similar in all periods, and BrdU positive cells were found on day 2. In rat molars, GIVE presented more positive cells for NG2 on day 2, for IdU on day 4 and CD90 on day 8. In the GIVC, CD90 was highly expressed in all periods analysed, the NG2 protein was also expressed in odontoblasts and CD90 in the subodontoblastic layer in rat molars. All mentioned proteins coexist in the human and animal pulp tissue forming heterogeneous perivascular and perineural cellular populations found in greater concentration in regions near the area of injury, suggesting that perivascular and perineural niches participate together in pulpal homeostasis in response to injury.

Selbstreproduktion ist eines der Kennzeichen aller lebenden Organismen. Der Zellzyklus dient der Selbstreproduktion in einzelligen Organismen. In mehrzelligen Organismen ist der Zellzyklus darüber hinaus für andere lebenswichtige Prozesse, einschließlich Immunreaktionen, unerlässlich. In dieser Arbeit wird eine Methode entwickelt mit der die Dauer der Zellzyklus Phasen bestimmt werden kann. Kenntnis über die Zellzyklusphasendauer ermöglicht vorherzusagen, wie schnell eine Population von proliferierenden Zellen wachsen wird, oder wie viele neue Zellen pro Stunde in einem Gewebe geboren werden. Im Kapitel 1 dieser Arbeit wird ein Zellzyklusmodell aufgestellt und mit experimentellen Bromdesoxyuridin Daten verglichen. Die Analyse zeigt, dass das Modell gut die experimentelle Kinetik beschreibt, hebt jedoch auch hervor dass einige der Parameter nicht identifiziert werden können. Dieses Problem wird in Kapitel 2 bearbeitet, wo zwei Ansätze erforscht werden, um den Informationsgehalt der Experimente zu erhöhen. In einem ersten Ansatz wird die Theorie der Versuchsplanung angewendet, um optimale Versuchspläne zu bestimmen. In einem zweiten Ansatz wird das übliche Bromdesoxyuridin Protokoll durch ein zweites Nukleosid erweitert. Beide Methoden verbessern in silico erheblich die Genauigkeit und Präzision der Abschätzungen. Im dritten Kapitel wird die Methodik in der Analyse der Keimzentrumsreaktion angewendet. Ein erheblicher Zufluss von Zellen in die dunkle Zone von Keimzentren wird vorhergesagt, und die Ansicht einer extrem schellen Zellteilung im Keimzentrum erscheint in dem Modell als ein Artefakt der Zellmigration. / Self-reproduction is one of the distinguishing marks of living organisms. The cell cycle is the underlying process by which self-reproduction is accomplished in single-celled organisms. In multi-cellular organisms, the cell cycle is in addition indispensable for other vital processes, including immune reactions. In this thesis a method is developed that allows to estimate the time it takes for a dividing cells to complete the CC phases. Knowledge of the CC phase durations allows to predict, for example, how fast a population of proliferating cells will grow in size, or how many new cells are born per hour in a given tissue. In Chapter 1 of this thesis, a cell cycle model with delays and variability in the completion times of each phase is developed. Analytical solutions are derived to describe a common experimental technique used for cell cycle analysis, namely pulse labeling with bromodeoxyuridine (BrdU). Comparison with data shows that the model reproduces closely measured cell cycle kinetics, however also reveals that some of the parameter values cannot be identified. This problem is addressed in Chapter 2. In a first approach, the framework of D-optimal experimental designs is employed, in order to choose optimal sampling schemes. In a second approach, the prevailing protocol with a single nucleoside is modified by adding a second nucleoside analog pulse. Both methods are tested and the results suggest that experimental design can significantly improve parameter estimation. In Chapter 3, the model is applied to the germinal center reaction. A substantial influx of cells into the dark zone of germinal centers is predicted. Moreover the wide-held view of rapid proliferation in germinal centers, appears, under this model, as an artifact of cell migration.

Obesity is a growing disorder in the United States, affecting over 60% of the population. We previously defined sympathetic nervous system (SNS) outflow from brain to white adipose tissue (WAT) using a viral transneuronal tract tracer. SNS innervation of WAT is the principle initiator of lipolysis, whereas decreases in sympathetic drive promote lipid accumulation. Which of the many origins of SNS outflow from brain to WAT results in SNS-mediated changes in lipid mobilization (increases in drive) or accumulation (decrease in drive) is unknown. Previous research indicates that sympathetic denervation blocks lipid mobilization; thus, rostral sites in the neuroaxis connected to WAT via the SNS may promote WAT lipid mobilization. The hypothalamic paraventricular nucleus (PVN) may play a role via its descending projections to the intermediolateral horn of the spinal cord. Therefore, the consequences of PVN lesions (PVNx) on WAT mobilization or accumulation were tested. PVNx resulted in increased lipid accumulation, indicated by increases in retroperitoneal (RWAT) , epididymal (EWAT) , and inguinal WAT (IWAT) pad masses, in fed hamsters, but PVNx did not block fasting (56 h)-induced lipid mobilization. Because adrenal medullary catecholamines, especially epinephrine, also play a minor role in lipid mobilization, we tested the contribution of catecholamine release on lipid mobilization through adrenal demedullation (ADMEDx), with and without PVNx, and found fastinginduced lipid mobilization was not blocked. There was, however, a suggestion that distal denervation of IWAT, with and without ADMEDx, partially blocked lipid mobilization. In addition, evidence suggests SNS also may be an important controller of fat cell proliferation. Surgical denervation of WAT triggers increases in fat cell number (FCN), but have not determined if this FCN increase is due to preadipocyte proliferation or differentiation of preadipocytes into mature fat cells. We also have not demonstrated what role sensory innervation may have in regulating white adipocyte proliferation. Therefore, the role of WAT sympathetic or sensory innervation on adipocyte proliferation was tested. The SNS but not sensory denervation triggered bona fide proliferation as indicated by bromodeoxyuridine plus AD3, a specific adipocyte membrane protein, colabeling. These and previous data suggest that the SNS plays a role in regulating adiposity.

Die Läsion des entorhinalen Kortex bei adulten Ratten induziert in der deafferenzierten Molekularschicht des Gyrus dentatus eine Gliaaktivierung und -proliferation. Histochemische Doppelfärbungen auf das astrozytenspezifische Antigen Glial fibrillary acidic protein oder den Mikrogliamarker Griffonia simplicifolia isolectin B4 und Bromodeoxyuridin haben gezeigt, daß die Mikrogliazellzahlen in der Molekularschicht des Gyrus dentatus 3 Tage nach Läsion (dpl) ein Maximum erreichten und 30 dpl auf Kontrollwerte zurückgingen. Die Astrozytenzahlen im ipsilateralen Gyrus dentatus erreichten 30 dpl ein Maximum, ihre größte Proliferationsaktivität war 7 dpl zu beobachten. 100 dpl waren die Astrozytenzahlen auf Kontrollwerte zurückgegangen. Die Gliaproliferation war nicht auf die ipsilaterale Molekularschicht beschränkt, sondern trat auch zu einem bestimmten Grad in der Körnerzellschicht und im kontralateralen Gyrus dentatus auf. Somit ruft eine entorhinale Kortexläsion eine rasche Mikrogliareaktion und eine langanhaltende Astrozytenaktivierung in der deafferenzierten Terminationszone des Tractus perforans hervor. Schließlich ist zu erwähnen, daß Gliaproliferation nach entorhinaler Läsion einem komplexen zeitlichen und räumlichen Muster folgt, das bei Prozessen der neuronalen und axonalen Reorganisation auftritt. / Entorhinal cortex lesion of adult rats induces glial activation and proliferation in the deafferented dentate molecular layer. Double-labelling immunocytochemistry for the astrocyte-specific antigen glial fibrillary acidic protein or the microglial cell marker Griffonia simplicifolia isolectin B4 with bromodexyuridine detection revealed that microglia counts and the proliferation rate in the ipsilateral dentate gyrus reached a maximum in the molecular layer at 3 days post-lesion (dpl) and returned to control levels by 30 dpl. Astrocyte counts in the ipsilateral dentate gyrus peaked at 30 dpl, with maximum proliferation at 7 dpl. At 100 dpl the astrocyte count had reverted to control levels. Glial proliferation was not restricted to the ipsilateral molecular layer but also occurred to some degree in the granule cell layer and the contralateral dentate gyrus. Thus entorhinal cortex lesion induces a rapid microglial reaction and long-lasting astrocyte activation in the deafferented termination zone of the perforant path. To conclude, glial proliferation after entorhinal cortex lesion follows a complex temporal and spatial pattern that coincides with processes of neuronal and axonal reorganization.

5-Bromo-2'-deoxyuridine (BrdU), a thymidine analogue, is genotoxic and teratogenic. The exposure of mouse embryos to BrdU at doses that cause malformations induces oxidative stress and an embryonic stress response characterized by an increase in c-Fos dependent AP-1 DNA binding. The goal of this thesis was to test the hypothesis that development is disturbed at sites where BrdU is incorporated into DNA, triggering oxidative stress and c-Fos induction. Gestation day 9 CD-1 mice were treated with BrdU and embryos were obtained for immunolocalization of BrdU, 8-oxoguanine, a biomarker for oxidative stress, and c-Fos. BrdU incorporation into DNA was dispersed throughout the embryo. In contrast, the staining for 8-oxoguanine and c-Fos were highest in the neuroepithelium. BrdU incorporation was not affected by the pre-administration of N-acetyl-cysteine (NAC), an anti-oxidant, although both 8-oxoguanine and c-Fos staining were decreased. Thus, the response of the embryo to insult is tissue specific.

No description available.