The antibodies against 5-bromo-2'-deoxyuridine specifically recognize trifluridine incorporated into DNA.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102 (also named TFTD), which consists of FTD and a thymidine phosphorylase inhibitor. FTD is supposed to exert its cytotoxicity via massive misincorporation into DNA, but the underlying mechanism of FTD incorporation into DNA and its correlation with cytotoxicity are not fully understood. The present study shows that several antibodies against 5-bromo-2'-deoxyuridine (BrdU) specifically cross-react with FTD, either anchored to bovine serum albumin or incorporated into DNA. These antibodies are useful for several biological applications, such as fluorescence-activated cell sorting, fluorescent immunostaining and immunogold detection for electron microscopy. These techniques confirmed that FTD is mainly incorporated in the nucleus during S phase in a concentration-dependent manner. In addition, FTD was also detected by immunohistochemical staining in paraffin-embedded HCT-116 xenograft tumors after intraperitoneal administration of FTD. Intriguingly, FTD was hardly detected in surrounding matrices, which consisted of fibroblasts with marginal expression of the nucleoside transporter genes SLC29A1 and SLC29A2. Thus, applications using anti-BrdU antibodies will provide powerful tools to unveil the underlying mechanism of FTD action and to predict or evaluate the efficacy and adverse effects of TAS-102 clinically.

Non-proliferating plasma cells detected in the salivary glands and bone marrow of autoimmune NOD.B10.H2b mice, a model for primary Sjögren's syndrome.

Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoimmune conditions such as primary Sjögren's syndrome (pSS). Particularly, the long-lived PC subset residing in the bone marrow has shown to continuously produce autoantibodies, whilst remaining unaffected by immunosuppressive treatment. We have previously shown accumulation of potentially long-lived PCs in chronically inflamed salivary glands of pSS patients. In this study, we aimed to characterise the PC compartment in the salivary glands (the target organ for pSS) and bone marrow before the onset of the murine pSS like disease versus advanced diseases progression. Bromodeoxyuridine (BrdU) was incorporated to distinguish the long-lived PCs. Double immunohistochemical staining and immunofluorescence were then conducted on submandibular gland and bone marrow sections from 8- and 40-week-old mice to identify BrdU and CD138. BrdU(+) cells were detected in the submandibular glands of 8-week-old mice, and observed within all focal infiltrates by 40 weeks of age. Most CD138(+) PCs were however BrdU(-) and located predominantly on the periphery of these infiltrates. This observation was verified through immunofluorescence. A comparable staining pattern was observed in the bone marrow of 8- and 40-week-old NOD.B10.H2b mice, where some of the CD138(+) cells also expressed BrdU. Interestingly, megakaryocytes in the bone marrow of NOD.B10.H2b mice were detected in close proximity to CD138(+) cells, illustrating a possible presence of PC survival niches. Our results demonstrate the presence and accumulation of potentially long-lived PCs in NOD.B10.H2b mice as the disease advances.

Development of a sensitive potency assay to measure the anti-proliferation effect of an anti-HER2 antibody.

For therapeutic antibodies that inhibit the growth of cancer cells, proliferation assays that measure cell number changes after the antibody treatment are often used to determine the potency of the antibody. Two of the most commonly used non-radioactive readout systems for proliferation assays, the ATP bioluminescence assay and the fluorescent dye Alamar Blue assay, were initially tested as potency assays an anti-HER2 antibody. Due to the slow growth of the target cells, these assays only produced less than 3-fold difference after 5 days of antibody treatment. BrdU incorporation-based proliferation assay, which differentiates proliferating cells from arrested cells, was developed, and showed superior sign-to-background ratio. Colorimetric, chemiluminescent, and DELFIA readouts were compared for BrdU incorporation assays, and DELFIA-based assay was further optimized using a Design of Experiment (DoE) approach. The final DELFIA-based BrdU incorporation assay demonstrated superior signal-to-background ratio, robustness, accuracy, and precision, and represented significant improvement over traditional proliferation assays.

Most anti-BrdU antibodies react with 2'-deoxy-5-ethynyluridine -- the method for the effective suppression of this cross-reactivity.

5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.

WNT/ß-catenin-signaling pathway stimulates the proliferation of cultured adult human Sertoli cells via upregulation of C-myc expression.

The role of WNT/ß-catenin-signaling pathway is critical in mouse Sertoli cell maturation and tumorigenesis. This study aims to examine the effects of WNT/ß-catenin signaling on the cultured adult human Sertoli cells and the underlying molecular mechanisms. Glycogen synthase kinase 3ß (GSK-3ß) inhibitors, SB216763 and lithium chloride (LiCl), were used to activate WNT/ß-catenin-signaling pathway. 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay and flow cytometry were used to analyze the proliferation and cell cycle of cultured human Sertoli cells, respectively. C-myc expression was accessed by immunofluorescence, real-time polymerase chain reaction and Western blot. The effects of c-myc on Sertoli cell proliferation were investigated by RNA interference technology and BrdU incorporation assay. The results showed activation of WNT/ß-catenin signaling stimulated human Sertoli cell proliferation. Obvious increases in c-myc messenger RNA and protein expression were observed after SB216763 and LiCl treatments. Knockdown of c-myc expression attenuated the ability of WNT/ß-catenin signaling to stimulate the proliferation of human Sertoli cells. WNT/ß-catenin signaling enhances human Sertoli cell proliferation via upregulation of c-myc expression.

Flow cytometry-based characterization of label-retaining stem cells following transplacental BrdU labelling.

A method to characterize and culture stem cells from neonate mouse epidermis after transplacental BrdU (bromo-deoxyuridine) administration is described. We have characterized stem cells by their properties viz. to retain BrdU label, adhere rapidly onto collagen-fibronectin substratum and express a specific biomarker beta-1-integrin. BrdU-labelled cells (detected using monoclonal antibody) constituted a sum of 18% of the total number of cells. The ability of freshly isolated keratinocytes [LRCs (label-retaining cells)] to bind to primary BrdU antibody or to pick up PI (propidium iodide) stain was distinguishable. Viable LRCs did not retain PI. Such cells, termed EpSC (epidermis stem cell), were PI negative and BrdU positive. EpSC constituted 6% of the total cell yield. Culture in low Ca2+ medium and susceptibility to differentiation in the presence of high Ca2+ levels further characterized the stem cells. This protocol is useful for studying transplacental carcinogenesis.

Radio-copper-labeled Cu-ATSM: an indicator of quiescent but clonogenic cells under mild hypoxia in a Lewis lung carcinoma model.

UNLABELLED: The purpose of this study is to reveal characteristics of (64)Cu-labeled diacetyl-bis(N(4)-methylthiosemicarbazone) ([(64)Cu]Cu-ATSM) during cell proliferation and hypoxia by autoradiography imaging and immunohistochemical staining. METHODS: The intratumoral distributions of [(64)Cu]Cu-ATSM and [(18)F]-2-fluoro-2-deoxy-D-glucose ([(18)F]FDG) in mice implanted with Lewis lung carcinoma (LLC1) tumor cells according to dual autoradiography were compared with the immunohistochemical staining patterns of proliferating markers [Ki-67 and 5-bromo-2'-deoxyuridine (BrdU)] and a hypoxic marker (pimonidazole). A clonogenic assay was performed using the cells of LLC1 tumor-implanted mice, and it was compared with the distribution of [(64)Cu]Cu-ATSM. RESULTS: [(64)Cu]Cu-ATSM mainly accumulated at the edge of tumors, whereas [(18)F]FDG was distributed inside the tumor and inside the [(64)Cu]Cu-ATSM accumulation. The number of Ki-67-positive cells/area tended to increase with [(18)F]FDG accumulation and decrease with [(64)Cu]Cu-ATSM accumulation. On the other hand, the number of BrdU-positive cells/area was negatively correlated with [(18)F]FDG accumulation and positively correlated with [(64)Cu]Cu-ATSM accumulation. High [(64)Cu]Cu-ATSM accumulation was found outside the high-[(18)F]FDG-accumulation and pimonidazole-positive regions. Colony formation ability was significantly higher in the tumor cells obtained from high-[(64)Cu]Cu-ATSM-accumulation regions than the cells from the intermediate- and the low-accumulation regions. CONCLUSION: [(64)Cu]Cu-ATSM accumulation regions in tumor cells indicate quiescent but clonogenic tumor cells under mild hypoxia. [(64)Cu]Cu-ATSM could play an important role in planning appropriate tumor radiotherapy.

Cell proliferation method: click chemistry based on BrdU coupling for multiplex antibody staining.

Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle (see UNIT 7.7). However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by an alkenyl deoxyuridine (5-ethynyl-2'-deoxyuridine, EdU), which is incorporated into DNA. The nucleotide exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is preferred over traditional BrdU detection since it is more sensitive and allows multicolor and multiplex analysis in FCM and imaging.

Improved methods for immunohistochemical detection of BrdU in hard tissue.

Bromodeoxyuridine (BrdU) is used to label synthesizing DNA and to chase label-retaining cell (LRC). As stem cells divide slowly in adult tissues, they can be visualized as LRCs. In order to identify LRCs in hard tissue, we examined optimal conditions of fixation, demineralization, and DNA denaturation/antigen retrieval for immunohistochemistry of BrdU in hard tissues including bone, tooth, and periodontal ligament. Mice were subcutaneously injected with BrdU (50 microg/g body weight) twice a day from the postnatal day 11 to day 15 and sacrificed at 2 h after the last injection. Dissected maxillae were fixed (Bouin's solution or 4% paraformaldehyde), demineralized (Morse's solution or EDTA), and embedded in paraffin. Antigen retrieval procedures were performed before incubation with primary antibody. When sections were treated with HCl for DNA denaturation, the staining intensity of BrdU positive cells was not affected by difference of fixatives. Higher sensitivity was obtained by demineralization with Morse than with EDTA. Although heat-induced antigen retrieval techniques in citrate buffer (pH 6.0) showed as well or better sensitivity than acid pretreatment, heating caused tissue damage specifically to tooth dentine and the surrounding tissue. When the LRCs at four weeks after the last injection of BrdU were compared, much more LRCs were observed in specimen demineralized with Morse than with 10% EDTA. Our data suggest that demineralization with Morse with Bouin fixative plus HCl pretreatment gives rise to the optimal results for BrdU immunodetection in hard tissue.

Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.

Use of halogenated precursors for simultaneous DNA and RNA detection by means of immunoelectron and immunofluorescence microscopy.

We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains.

Combined bromodeoxyuridine immunocapture and terminal-restriction fragment length polymorphism analysis highlights differences in the active soil bacterial metagenome due to Glomus mosseae inoculation or plant species.

High numbers of bacteria are associated with arbuscular mycorrhizal (AM) fungi, but their functions and in situ activities are largely unknown and most have never been characterized. The aim of the present study was to study the impact of Glomus mosseae inoculation and plant type on the active bacterial communities in soil by using a molecular approach, bromodeoxyuridine (BrdU) immunocapture in combination with terminal-restriction fragment length polymorphism (T-RFLP). This approach combined with sequence information from clone libraries, enabled the identification of actively growing populations, within the total bacterial community. Distinct differences in active bacterial community compositions were found according to G. mosseae inoculation, treatment with an antifungal compound (Benomyl) and plant type. The putative identities of the dominant bacterial species that were activated as a result of G. mosseae inoculation were found to be mostly uncultured bacteria and Paenibacillus species. These populations may represent novel bacterial groups that are able to influence the AM relationship and its subsequent effect on plant growth.

Maintenance of long-term tumour-specific T-cell memory by residual dormant tumour cells.

LacZ (Gal)-reactive immune cells were transferred into athymic nu/nu mice inoculated with Gal-expressing syngeneic tumour cells (ESbL-Gal) in order to study tumour-protective T-cell memory. This transfer prevented tumour outgrowth in recipients and resulted in the persistence of a high frequency of Gal-specific CD8(+) T cells in the bone marrow and spleen. In contrast, such Ag-specific memory CD8(+) T cells were not detectable by peptide-major histocompatibility complex (MHC) multimer staining in animals that had not previously received an antigenic challenge. Even though CD44(hi) memory T cells from the bone marrow showed a significantly higher turnover rate, as judged by bromodeoxyuridine (BrdU) incorporation, than respective cells from spleen or lymph nodes, as well as in comparison to CD44(lo) naïve T cells, these findings suggest that tumour-associated antigen (TAA) from residual dormant tumour cells are implicated in maintaining high frequencies of long-term surviving Gal-specific memory CD8(+) T cells. Memory T cells could be recruited to the peritoneal cavity by tumour vaccination of immunoprotected nu/nu mice and exhibited ex vivo antitumour reactivity. Long-term immune memory and tumour protection could be maintained over four successive transfers between tumour-inoculated recipients, which involved periodic antigenic restimulation in vivo prior to reisolating the cells for adoptive transfer. Using a cell line (ESbL-Gal-BM) that was established from dormant tumour cells isolated from the bone marrow of immunoprotected animals, it could be demonstrated that the tumour cells had up-regulated the expression of MHC class I molecules and down-regulated the expression of several adhesion molecules during the in vivo passage. Our results suggest that the bone marrow microenvironment has special features that are of importance for the maintenance of tumour dormancy and immunological T-cell memory, and that a low level of persisting antigen favours the maintenance of Ag-specific memory T cells over irrelevant memory T cells.

Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus.

Cell proliferation can be detected by the incorporation of tritiated thymidine (3H-dT) or halopyrimidines during DNA synthesis in progenitor cells. Administration of two thymidine analogues at different times can further determine the cell-cycle kinetics of proliferating cells. Traditionally, this was done by combining bromodeoxyuridine (BrdU) immunocytochemistry and 3H-dT autoradiography, or by BrdU and iododeoxyuridine (IdU) double-labeling using two mouse antibodies. However, these methods either require lengthy exposure time or involve complicated histological procedures for differentiating between two antibodies of the same species. Here we report a simple and reliable method of distinguishing BrdU- and IdU-labeled cells by immunofluorescence. This method uses a mouse monoclonal antibody that recognizes both BrdU and IdU and a rat anti-BrdU antibody that has no cross-reactivity with IdU. When combined with species-specific secondary antibodies that are conjugated to different fluorophores, this method identifies BrdU- and IdU-incorporation as doubly and singly labeled cells, respectively. This method has broad applications. First, we demonstrate that this method can distinguish mouse cortical neurons generated on different embryonic days. Second, by administering IdU and BrdU at varying intervals, we used this method to calculate that the length of S-phase of neural progenitor cells in the adult mouse dentate gyrus is approximately 6 h. Finally, we show that a six-fold higher concentration of IdU detects only 10% more cells than the standard dose of BrdU (50 mg/kg) using the double-labeling method. These results suggest that the standard dose of BrdU is sufficient to label the majority of proliferative populations in the S-phase in pulse labeling experiments.

Autoantibodies to REG, a beta-cell regeneration factor, in diabetic patients.

BACKGROUND: Regenerating gene (Reg) product, Reg, acts as an autocrine/paracrine growth factor for beta-cell regeneration. The presence of autoimmunity against REG may affect the operative of the regenerative mechanisms in beta cells of Type 1 and Type 2 diabetes patients. We screened sera from Type 1 and Type 2 diabetes subjects for anti-REG autoantibodies, searched for correlations in the general characteristics of the subjects with the presence of anti-REG autoimmunity, and tested the attenuation of REG-induced beta-cell proliferation by the autoanitibodies. MATERIAL AND METHODS: We examined the occurrence of anti-REG autoantibodies in patients' sera (265 Type 1, 368 Type 2 diabetes patients, and 75 unrelated control subjects) by Western blot analysis, and evaluated inhibitory effects of the sera on REG-stimulated beta-cell proliferation by a 5'-Bromo-2'-deoxyuridine (BrdU) incorporation assay in vitro. RESULTS: Anti-REG autoantibodies were found in 24.9% of Type 1, 14.9% of Type 2 and 2.7% of control subjects (P = 0.0004). There were significant differences between the autoantibody positive and negative groups in the duration of disease in the Type 1 subjects (P = 0.0035), and the age of onset in the Type 2 subjects (P = 0.0274). The patient sera containing anti-REG autoantibodies significantly attenuated the BrdU incorporation by REG (35.6 +/- 4.06% of the control), whereas the nondiabetic sera without anti-REG autoantibodies scarcely reduced the incorporation (88.8 +/- 5.10%). CONCLUSION: Anti-REG autoantibodies, which retard beta-cell proliferation in vitro, are found in some diabetic patients. Thus, autoimmunity to REG may be associated with the development/acceleration of diabetes in at least some patients.

Marrow stromal cell transplantation after traumatic brain injury promotes cellular proliferation within the brain.

OBJECTIVE: This study was designed to investigate the effects of intracerebral as well as intravenous administration of bone marrow stromal cells (MSCs) on endogenous cellular proliferation after traumatic brain injury (TBI). METHODS: Two experimental groups of Wistar rats were studied. One group received MSCs intracerebrally, and the other group received MSCs intravenously after injury by controlled cortical impact. MSCs were harvested from the bone marrow of male Wistar rats. For the intracerebral study, 24 male rats were divided into three groups (eight rats per group): rats injected with MSCs (1 x 10(6)) intracerebrally 1 day after TBI; 2) rats injected with phosphate-buffered saline intracerebrally 1 day after TBI; and 3) sham group not subjected to injury and not administered treatment. For the intravenous study, 10 female Wistar rats were injected 1 day after TBI with either MSCs (2 x 10(6)) (n = 5) or phosphate-buffered saline (n = 5) via the tail vein. Neurological function of the rats was evaluated with modified neurological severity scores and rotarod motor test. All rats were injected with bromodeoxyuridine intraperitoneally, to label the newly generating cells. Rats were killed 15 days after TBI, and coronal brain sections were stained immunohistochemically with diaminobenzidine to identify newly generating bromodeoxyuridine-positive cells. To study the differentiation of newly generating cells into neurons, sections were also double-stained for neuronal markers (Tuj1, doublecortin, NeuN) with fluorescein isothiocyanate. RESULTS: The data demonstrate that newly generating cells were mainly present in the subventricular zone, hippocampal formation, and boundary zone of contusion of both treated and control animals. Intracerebral MSC treatment significantly increased the progenitor cell proliferation in the subventricular zone and boundary zone compared with the controls, whereas intravenous MSC treatment enhanced this endogenous proliferation in subventricular zone, hippocampus, and boundary zone. In both groups, some of the new cells revealed positive staining for neuronal markers. A statistically significant functional improvement was observed in both the intracerebrally as well as intravenously treated groups. CONCLUSION: Intracerebral and intravenous MSC administration promotes endogenous cellular proliferation after TBI in rats. This may contribute to the functional improvement observed in these rats.

Immunocytochemical identification of neoplastic cell clone in phase S of cell cycle in transplantable rat tumors.

Wistar and Buffalo rats of both sexes, aged 4 months, were divided into three groups: I which was given an intramuscular injection of 3 x 10(6) cells of Morris hepatoma (Buffalo males), II--subcutaneous injection of 3 x 10(4) cells of mammary gland carcinoma (Wistar females), III--intraperitoneal injection of 3 x 10(4) cells of Yoshid sarcoma (Wistar males). The animals were killed: in group I--19, group II--13 and in group III--6 days after tumor transplantation. Twenty four hours before euthanasia the rats were given 5-brome-2'-deoxyuridine (BRd-U) at a dose of 50 mg/kg body mass. The control group consisted of animals with tumour. They were not treated with BRd-U. Immunocytochemical reaction was performed on the sections of tumors, using monoclonal anti-BRd-U clone BU-33, Sigma. Computer measurements of tumor cells were carried out. There was a high similarity in morphological parameters between two kinds of cancer, and clear differences between them and Yoshid sarcoma. The main difference was noted in a twofold increase in the quantity of synthesised DNA in the nuclei of sarcoma cells. Immunocytochemical identification of tumor cells in phase S of the cell cycle with the use of monoclonal anti-BRd-U antibody is a precise and quick method of estimation of their proliferative potential.

Neuropeptide Y promotes beta-cell replication via extracellular signal-regulated kinase activation.

AIMS/HYPOTHESIS: Previous studies have shown that neuropeptide Y (NPY) gene expression and release are increased in hyperphagic ob/ob mice and diabetic rats. Therefore, we hypothesized that orexigenic agent, NPY, has the effect on the obesity and diabetes. To elucidate the relationship, we have studied the regulatory role of NPY on islet cells. METHODS: Isolated islets were incubated with NPY or NPY Y1 receptor specific antagonist, BIBP3226. Proliferation, apoptosis, and Y1 receptor expression were identified by immunohistochemistry. We studied that ERK1/2 mediates the NPY pathway with PD98059 (MAP kinase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), and BIM-1 (protein kinase C inhibitor). After NPY-treated islets were exposed to high glucose, insulin levels were detected. RESULTS: beta-Cell replication was enhanced in a dose-dependent manner, but without any changes on the other cells in islet. NPY Y1 receptors were expressed on islet and NPY induced phosphorylation of ERK1/2 rapidly and transiently. PD98059 (MAPK kinase inhibitor) and BIM-1 (protein kinase C inhibitor) inhibited activation of ERK1/2 by NPY, but wortmannin (phosphatidylinositol 3-kinase inhibitor) did not. Exposure of NPY-treated islets to high glucose showed the decreasing trend of insulin secretion. CONCLUSION/INTERPRETATION: Our data suggest that NPY promotes beta-cell replication via extracellular signal-regulated kinase activation and inhibits glucose-stimulated insulin secretion.

Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.

Arbuscular mycorrhizae are beneficial for crops grown under low-till management systems. Increasingly, it is becoming apparent that bacteria associated with mycorrhizae can enhance the beneficial relationship between mycorrhizae and plants. However, it has been difficult to study these relationships by conventional techniques. In this study actively growing bacteria were identified in soil from an undisturbed fallow field known to contain arbuscular mycorrhizae by using molecular tools to eliminate the need for cultivation. A thymidine analog, bromodeoxyuridine (BrdU), was added to the soil and incubated for 2 days. DNA was extracted, and the newly synthesized DNA was isolated by immunocapture of the BrdU-containing DNA. The active bacteria in the community were identified by 16S rRNA gene PCR amplification and DNA sequence analysis. Based on 16S rRNA gene sequence information, a selective medium was chosen to isolate the corresponding active bacteria. Bacillus cereus strain VA1, one of the bacteria identified by the BrdU method, was isolated from the soil and tagged with green fluorescent protein. By using confocal microscopy, this bacterium was shown to clearly attach to arbuscular mycorrhizal hyphae. This study was the first to use this combination of molecular and traditional approaches to isolate, identify, and visualize a specific bacterium that is active in fallow soil and associates with arbuscular mycorrhizal hyphae.