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Experimental Biology and Medicine... Sep 2017Intestinal epithelial tissue is constantly regenerated as a means to maintain proper tissue function. Previous studies have demonstrated that denervation of the...
Intestinal epithelial tissue is constantly regenerated as a means to maintain proper tissue function. Previous studies have demonstrated that denervation of the parasympathetic or sympathetic nervous system to the intestine alters this process. However, results are inconsistent between studies, showing both increases and decreases in proliferation after denervation of the parasympathetic or sympathetic. The effect appears to correlate with (1) the timing post-denervation, (2) denervation-induced changes in food intake, (3) the denervation technique used, and (4) which intestinal segment is investigated. Thus, we proposed that parasympathetic or sympathetic denervation does not have an effect on intestinal epithelial regeneration when you (1) evaluate denervation after long-term denervation, (2) control for post-surgical changes in food intake, (3) use minimally invasive surgical techniques and (4) include a segmental analysis. To test this, adult male Sprague Dawley rats underwent parasympathetic denervation via subdiaphragmatic vagotomy, sympathetic denervation via celiacomesenteric ganglionectomy, a parasympathetic denervation sham surgery, or a sympathetic denervation sham surgery. Sham surgery ad libitum-fed groups and sham surgery pair-fed groups were used to control for surgically induced changes in food intake. Three weeks post-surgery, animals were sacrificed and tissue from the duodenum, jejunum, and ileum was excised and immunohistochemically processed to visualize indicators of proliferation (bromodeoxyuridine-positive cells) and apoptosis (caspase-3-positive cells). Results showed no differences between groups in proliferation, apoptosis, or total cell number in any intestinal segment. These results suggest that parasympathetic or sympathetic denervation does not have a significant long-term effect on intestinal epithelial turnover. Thus, intestinal epithelial regeneration is able to recover after autonomic nervous system injury. Impact statement This study investigates the long-term effect of autonomic denervation on intestinal epithelial cell turnover, as measured by proliferation, apoptosis, and total cell number. Although previous research has established that autonomic denervation can alter intestinal epithelial turnover under short-term conditions, here we establish for the first time that these changes do not persist long-term when you control for surgical-induced changes in food intake and use targeted denervation procedures. These findings add to the base of knowledge on autonomic control of tissue turnover, highlight the ability of the intestinal epithelium to recover after autonomic injury and reveal possible implications of the use of ANS denervation for disease treatment in humans.
Topics: Animals; Apoptosis; Bromodeoxyuridine; Caspase 3; Cell Proliferation; Epithelial Cells; Immunohistochemistry; Intestine, Small; Male; Parasympathectomy; Rats, Sprague-Dawley; Sympathectomy; Time
PubMed: 28766984
DOI: 10.1177/1535370217724790 -
Cytometry Nov 1985This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this...
This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.
Topics: Animals; Antibodies, Monoclonal; Bromodeoxyuridine; Cell Line; Cricetinae; Cricetulus; DNA; DNA Replication; DNA, Single-Stranded; Hot Temperature; Humans; Nucleic Acid Denaturation; Propidium
PubMed: 4064837
DOI: 10.1002/cyto.990060606 -
Cell Proliferation Apr 2013Previous studies have shown alterations in bone marrow cell proliferation in malnourished rats, during lactation. The objective of this study was to determine in vivo...
OBJECTIVES
Previous studies have shown alterations in bone marrow cell proliferation in malnourished rats, during lactation. The objective of this study was to determine in vivo effects of moderate and severe malnutrition on spleen cell proliferation in 21-day-old rat pups.
MATERIALS AND METHODS
Spleen cell proliferation was determined following administration of bromodeoxyuridine (BrdUrd) over a time course of 2, 4, 6 and 8 h. Incorporation of BrdUrd was detected using FITC-conjugated anti-BrdUrd monoclonal antibodies and total DNA content was detected and evaluated using propidium iodide using flow cytometry.
RESULTS
Proportions of cells in S and G2 /M were reduced in the rats with moderate (MN2(nd) ) and severe (MN3(rd) ) malnutrition. BrdUrd incorporation was lower in both groups of malnourished rat. In cells of MN2nd individuals, length of G1 became shorter, while length of S-phase increased. In contrast, fraction of cells in proliferation was significantly lower in both groups of malnourished rat, with MN3rd group having lowest percentage of cell population growth. In this study, severe malnutrition did not significantly affect duration of phases of the cell cycle, although fractions of proliferating cells were dramatically reduced.
CONCLUSION
Moderate malnutrition increased time of cells in DNA synthesis and time of total cell cycle and severe malnutrition reduced growth fraction of spleen cells in malnourished rats during lactation.
Topics: Animals; Antibodies, Monoclonal; Body Weight; Bromodeoxyuridine; Cell Count; Cell Cycle; Cell Proliferation; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; Lactation; Malnutrition; Organ Size; Propidium; Rats; Rats, Wistar; Spleen; Time Factors; Weaning
PubMed: 23510471
DOI: 10.1111/cpr.12019 -
Uirusu Dec 1987
Review
Topics: Antiviral Agents; Bromodeoxyuridine; Herpes Zoster; Herpesvirus 3, Human; Humans
PubMed: 2833837
DOI: 10.2222/jsv.37.179 -
Journal of Immunological Methods Mar 2022Quantitative detection of T cell proliferation is an important readout in immunology research, as it is one of the hallmarks of T cell activation. Fluorescence-based...
Quantitative detection of T cell proliferation is an important readout in immunology research, as it is one of the hallmarks of T cell activation. Fluorescence-based methods for T cell proliferation mostly rely on the usage of probes that non-specifically conjugate to free primary amine groups in cells. Each cell division then results in a two-fold dilution of the probes which is detectable with flow cytometry. However, questions have been raised about cytotoxicity of these dilution-based T cell proliferation probes and they potentially affect T cell activation. An alternative assay relies on the incorporation of the uridine analog BrdU in the DNA of dividing T cells that can be detected with an antibody, but this requires harsh fixation and denaturation conditions. Recently, a new assay for detection of cell proliferation has been developed, based on the incorporation of the bioorthogonally-functionalized uridine analog 5-ethynyl-2'-deoxyuridine (EdU). Goal of this study was to compare the sensitivity and cytotoxicity of the EdU assay with a widely-used dilution-based T cell proliferation probe, CellTrace Far Red. We found that, compared to the dilution-based probe, the EdU-based assay better preserves T cell viability, is more sensitive for detecting T cell proliferation, and allows for better discernable interferon gamma responses.
Topics: Antineoplastic Agents; Bromodeoxyuridine; Cell Proliferation; Deoxyuridine; Flow Cytometry; Staining and Labeling; T-Lymphocytes; Uridine
PubMed: 35074315
DOI: 10.1016/j.jim.2022.113228 -
Journal of Visualized Experiments : JoVE Sep 2011Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial...
Bromodeoxyuridine (BrdU) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton.
Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS). Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.
Topics: Bacteria; Bromodeoxyuridine; Carbon; Flow Cytometry; Plankton; RNA, Ribosomal, 16S; Water; Water Microbiology
PubMed: 21931294
DOI: 10.3791/2855 -
Histochemistry and Cell Biology Feb 2022Detection of synthetic thymidine analogues after their incorporation into replicating DNA during the S-phase of the cell cycle is a widely exploited methodology for...
Detection of synthetic thymidine analogues after their incorporation into replicating DNA during the S-phase of the cell cycle is a widely exploited methodology for evaluating proliferative activity, tracing dividing and post-mitotic cells, and determining cell-cycle parameters both in vitro and in vivo. To produce valid quantitative readouts for in vivo experiments with single intraperitoneal delivery of a particular nucleotide, it is necessary to determine the time interval during which a synthetic thymidine analogue can be incorporated into newly synthesized DNA, and the time by which the nucleotide is cleared from the blood serum. To date, using a variety of methods, only the bioavailability time of tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) have been evaluated. Recent advances in double- and triple-S-phase labeling using 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU), and 5-ethynyl-2'-deoxyuridine (EdU) have raised the question of the bioavailability time of these modified nucleotides. Here, we examined their labeling kinetics in vivo and evaluated label clearance from blood serum after single intraperitoneal delivery to mice at doses equimolar to the saturation dose of BrdU (150 mg/kg). We found that under these conditions, all the examined thymidine analogues exhibit similar labeling kinetics and clearance rates from the blood serum. Our results indicate that all thymidine analogues delivered at the indicated doses have similar bioavailability times (approximately 1 h). Our findings are significant for the practical use of multiple S-phase labeling with any combinations of BrdU, IdU, CldU, and EdU and for obtaining valid labeling readouts.
Topics: Animals; Biological Availability; Bromodeoxyuridine; Dentate Gyrus; Deoxyuridine; Glyburide; Injections, Intraperitoneal; Kinetics; Mice; Mice, Inbred C57BL; Thymidine
PubMed: 34757474
DOI: 10.1007/s00418-021-02048-y -
Bioorganic & Medicinal Chemistry Letters Dec 2016Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in terms of cellular uptake, nucleoside kinase-mediated activation...
Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in terms of cellular uptake, nucleoside kinase-mediated activation and catabolism are well-documented. The monophosphate pro-nucleotides known as ProTides represents a powerful strategy for bypassing the dependence on active transport and nucleoside kinase-mediated activation. Herein, we report the structural tuning of BVdU ProTides. Forty six phosphoramidates were prepared and biologically evaluated against three different cancer cell lines; murine leukemia (L1210), human CD T-lymphocyte (CEM) and human cervical carcinoma (HeLa). Twenty-fold potency enhancement compared to BVdU was achieved against L1210 cells. Interestingly, a number of ProTides showed low micromolar activity against CEM and HeLa cells compared to the inactive parent BVdU. The ProTides showed poor, if any measurable toxicity to non-tumourigenic human lung fibroblast cell cultures. Separation of four pairs of the diastereoisomeric mixtures and comparison of their spectral properties, biological activities and enzymatic activation rate is reported.
Topics: Amides; Animals; Antineoplastic Agents; Antiviral Agents; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Humans; Mice; Neoplasms; Phosphoric Acids
PubMed: 27818111
DOI: 10.1016/j.bmcl.2016.10.077 -
Molecular & Cellular Proteomics : MCP Jan 2003The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report,...
The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2'-deoxyuridine. A key question regarding the use of photoaptamer probes is the specificity of the cross-linking reaction. The specificity of three photoaptamers was explored by comparing their reactions with target proteins and non-target proteins. The range of target/non-target specificity varies from 100- to >10(6)-fold with most values >10(4)-fold. The contributions of the initial binding step and the photocross-linking step were evaluated for each reaction. Photocross-linking never degraded specificity and significantly increased aptamer specificity in some cases. The application of photoaptamer technology to proteomics was investigated in microarray format. Immobilized anti-human immunodeficiency virus-gp120 aptamer was able to detect subnanomolar concentrations of target protein in 5% human serum. The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology.
Topics: Bromodeoxyuridine; Cross-Linking Reagents; Escherichia coli; HIV Envelope Protein gp120; Kinetics; Oligonucleotide Array Sequence Analysis; Peptides; Protein Array Analysis; Protein Binding; Proteomics; Sensitivity and Specificity
PubMed: 12601078
DOI: 10.1074/mcp.m200059-mcp200 -
Brain Structure & Function Sep 2018The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, which may trigger vascular thrombosis with...
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, which may trigger vascular thrombosis with consecutive infarcts. However, cognitive dysfunctions representing one of the most commonest neuropsychiatric symptoms are frequently present despite the absence of any ischemic brain lesions. Data on the structural and functional basis of the neuropsychiatric symptoms are sparse. To examine the effect of APS on hippocampal neurogenesis and on white matter, we induced experimental APS (eAPS) in adult female Balb/C mice by immunization with β2-glycoprotein 1. To investigate cell proliferation in the dentate gyrus granular cell layer (DG GCL), eAPS and control mice (n = 5, each) were injected with 5-bromo-2'-deoxyuridine (BrdU) once a day for 10 subsequent days. Sixteen weeks after immunization, eAPS resulted in a significant reduction of BrdU-positive cells in the DG GCL compared to control animals. However, double staining with doublecortin and NeuN revealed a largely preserved neurogenesis. Ultrastructural analysis of corpus callosum (CC) axons in eAPS (n = 6) and control mice (n = 7) revealed no significant changes in CC axon diameter or g-ratio. In conclusion, decreased cellular proliferation in the hippocampus of eAPS mice indicates a limited regenerative potential and may represent one neuropathological substrate of cognitive changes in APS while evidence for alterations of white matter integrity is lacking.
Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Autoantigens; Behavior Rating Scale; Bromodeoxyuridine; Cell Differentiation; Cell Proliferation; Corpus Callosum; Dentate Gyrus; Disease Models, Animal; Female; Fluorescence; Mice; Mice, Inbred BALB C; Neurogenesis; Radiation-Sensitizing Agents; beta 2-Glycoprotein I
PubMed: 29936552
DOI: 10.1007/s00429-018-1699-9