scholarly journals Quantitative Analysis of CBP- and P300-Induced Histone Acetylations In Vivo Using Native Chromatin

2003 ◽  
Vol 23 (21) ◽  
pp. 7611-7627 ◽  
Author(s):  
Kirk J. McManus ◽  
Michael J. Hendzel
Keyword(s):  
Substrate Specificity ◽  
Histone Deacetylases ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Resolving Power ◽  
Creb Binding Protein ◽  
Histone Tails ◽  
Nonhistone Proteins

ABSTRACT In vivo, histone tails are involved in numerous interactions, including those with DNA, adjacent histones, and other, nonhistone proteins. The amino termini are also the substrates for a number of enzymes, including histone acetyltransferases (HATs), histone deacetylases, and histone methyltransferases. Traditional biochemical approaches defining the substrate specificity profiles of HATs have been performed using purified histone tails, recombinant histones, or purified mononucleosomes as substrates. It is clear that the in vivo presentation of the substrate cannot be accurately represented by using these in vitro approaches. Because of the difficulty in translating in vitro results into in vivo situations, we developed a novel single-cell HAT assay that provides quantitative measurements of endogenous HAT activity. The HAT assay is performed under in vivo conditions by using the native chromatin structure as the physiological substrate. The assay combines the spatial resolving power of laser scanning confocal microscopy with simple statistical analyses to characterize CREB binding protein (CBP)- and P300-induced changes in global histone acetylation levels at specific lysine residues. Here we show that CBP and P300 exhibit unique substrate specificity profiles, consistent with the developmental and functional differences between the two HATs.

The Growth Inhibition of Targeted Pluronic/Formononetin Nanocomposite on Oral Squamous Cell Carcinoma In Vitro

2020 ◽  
Vol 12 (8) ◽  
pp. 1022-1029
Author(s):  
Ming Liu ◽  
Chen Lin ◽  
Xiaoqing Huang ◽  
Yuxiang Lin
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Growth Inhibition ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Laser Scanning ◽  
Critical Concentration ◽  
Laser Scanning Confocal Microscopy

Natural flavonoid formononetin (FN) has anticancer effects, but the hydrophobic structure, characteristics of the short half-life in vivo, limiting its clinical wide-ranging application. In this study, FN loaded Pluronic (PF)@folic acid (FA) micelles (FN-PF@FA), were prepared to improve the solubility, bioavailability and targeting. FA coupling PF was prepared by carbodiimide crosslinker chemical method, FN-PF@FA micelles were prepared by modified film hydration method, and compared the antitumor activity of FN loaded micelles with free FN In Vitro. The spherical smooth surface of FN-PF@FA micelles had smaller particle size (112.3±5.3 nm), high encapsulation efficiency (86.14±2.68%), high negative zeta potential (-25.8±0.57 mV), low critical concentration CMC (0.03 mg/mL), and better sustained release profile. In addition, FN-PF@FA micelles have a positive targeting effect on oral squamous cell carcinoma cells (SCC3). In 48 hours, the growth inhibition of 50% (GI50) was 28.6±1.2 μg/mL for FN and 17.4±0.78 μg/mL for FN-PF, the dose dropped by nearly 38.46%. In addition, the GI50 value of FN-PF@FA was 9.5±0.3 μg/mL, 66.43% lower than FN and 44.83% lower than FN-PF. Furthermore, the laser scanning confocal microscopy revealed that the conjugation of FA significantly improves the active targeting ability of micelles. FN-PF@FA micelles have the potential to target the release of anticancer drugs with higher bioavailability, further provides a new avenue for the application of traditional Chinese medicine extract in oral malignant tumor.

2,3′,4,4′,5-Pentachlorobiphenyl Induced Thyrocyte Autophagy by Promoting Calcium Influx via Store-Operated Ca2+ Entry

2020 ◽  
Vol 177 (2) ◽  
pp. 483-493
Author(s):  
Li Wang ◽  
Wenli Xu ◽  
Qi Zhou ◽  
Bojin Xu ◽  
Yunlu Sheng ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Calcium Influx ◽  
Underlying Mechanism ◽  
Laser Scanning Confocal Microscopy ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Class Iii ◽  
Rat Thyroid

Abstract PCB118, a 2,3′,4,4′,5-pentachlorobiphenyl, has been shown to destroy thyroidal ultrastructure and induce thyrocyte autophagy. Previously, we reported that PCB118 promoted autophagosome formation in vivo and in vitro, but more details remain to be revealed. To explore the underlying mechanism by which PCB118 regulates thyrocyte autophagy, Fischer rat thyroid cell line-5 (FRTL-5) cells were exposed to different doses of PCB118 at 0, 0.25, 2.5, and 25 nM for 0–48 h. Western blot analysis of autophagy-related proteins P62, BECLIN1, and LC3 demonstrated that PCB118 induced autophagy formation in dose- and time-dependent manner. Moreover, laser scanning confocal microscopy and flow cytometry showed PCB118 treatment led to time- and dose-dependent increase in intracellular calcium concentration ([Ca2+]i). Additionally, PCB118 promoted store-operated Ca2+ entry (SOCE) channel followed by significant increase of ORAI1 and STIM1 protein levels. On the other hand, PCB118 induced thyroidal autophagy via class III β-tubulin (TUBB3)/death-associated protein kinase 2 (DAPK2)/myosin regulatory light chain (MRLC)/autophagy-related 9A (ATG9A) pathway in FRTL-5 cells. Pretreatment with SOCE inhibitor SKF96365 reduced cytosolic Ca2+, ORAI1, STIM1, and BECLIN1 levels as well as LC3 II/LC3 I ratio, while increased P62 expression. SKF96365 also inhibited TUBB3/DAPK2/MRLC/ATG9A pathway in FRTL-5 cells treated by PCB118. Our results provide evidence that PCB118 may induce thyroidal autophagy through TUBB3-related signaling pathway, and these effects are likely to be regulated by calcium influx via SOCE channel.

scholarly journals Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System

2021 ◽  
Vol 12 ◽  
Author(s):  
Ping He ◽  
Shu Li ◽  
Shengtao Xu ◽  
Huacai Fan ◽  
Yongfen Wang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Transformation System ◽  
Scanning Confocal Microscopy ◽  
Bacillus Spp ◽  
Green Fluorescent ◽  
Race 4

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.

68 TRANSCRIPTIONAL GENOME ACTIVATION IN CANINE EMBRYOS COLLECTED IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 146 ◽  
Author(s):  
S. Chastant-Maillard ◽  
C. Viaris de Lesegno ◽  
S. Thoumire ◽  
M. Chebrout ◽  
K. Reynaud
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
Staining Intensity ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Stage ◽  
Morula Stage

Early embryonic stages are supported by maternal transcripts from the oocyte cytoplasm. Progressive transcription of embryonic genome is a key step for further embryonic development, especially during in vitro culture. To date, in vitro culture from fertilization to the blastocyst stage is inefficient in the canine species. The objective of this work was to identify minor and major activation in in vivo-produced dog embryos. Ovariectomies were performed in 31 Beagle bitches from 102 to 266 h after ovulation (post-ov), precisely timed by transabdominal ultrasonography. Embryos were collected by tubal flushing with M199-Hepes and immediately transferred into transcription buffer. Transcriptional activity was evaluated through 5-bromouridine 5′-triphosphate (BrUTP) incorporation in nascent RNA, without microinjection (Aoki et al. 1997). Oocytes from anoestrus ovaries were used as positive controls. 5-Bromouridine 5′-triphosphate incorporation was revealed by immunocytochemistry (anti-bromodeoxyuridine primary antibody) and embryonic DNA was stained by ethidium homodimer-2. Staining was quantified under laser scanning confocal microscopy. Transcriptional activity was calculated as (mean nuclear intensity – cytoplasmic mean intensity) × nuclear area and expressed in arbitrary units (AU). It was compared to 1 (similar intensity in nucleus and cytoplasm; i.e. no transcriptional activity) by t-test; levels of transcriptional activity were compared between stages by variance analysis. Seventy embryos (from 7 to 21 per stage) from 31 bitches were analysed, from 2 pronuclei to morula stage. Between 28 and 125 nuclei were quantified per stage. At each stage, transcriptional activity was calculated per embryo and per nucleus. A significant transcriptional activity was detected as early as the 2 pronuclei stage (102–132 h post-ov; 1.15 ± 0.05 AU). Transcriptional activity per embryo significantly increased between the 2- and the 4-cell stage and between the 8-cell and the morula stage. In early 8-cell embryos, staining intensity of the various nuclei was markedly heterogeneous within the same embryo, all nuclei being intensively stained from the late 8-cell stage onwards. Transcriptional activity per nucleus increased also from the 2- to the 4-cell stage (respectively, 120–161 h post-ov, 1.15 ± 0.02 AU and 133–154 h post-ov, 1.35 ± 0.04 AU) until the 8-cell stage (153–225 h post-ov, 5.12 ± 0.55 AU). Transcriptional levels at these 3 stages differed significantly. It decreased between the 8-cell and the morula stage (230–266 h post-ov, 3.06 ± 0.13 AU), probably reflecting the acquisition of a selectivity in gene expression at major activation, as in other species; Nothias et al. 1995). Addition of the transcriptional inhibitor α-amanitin during BrUTP incubation decreased the transcriptional activity by 60% (P < 0.05). Embryonic gene expression (minor activation) thus begins in the canine embryo as early as the 2 pronuclei stage, with major activation taking place during the 8-cell stage.

scholarly journals Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Brain Barrier ◽  
Scanning Confocal Microscopy ◽  
Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

scholarly journals Integrin αvβ6-targeted MR molecular imaging of breast cancer in a xenograft mouse model

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dengfeng Li ◽  
Chengyan Dong ◽  
Xiaohong Ma ◽  
Xinming Zhao
Keyword(s):  
Breast Cancer ◽  
Molecular Imaging ◽  
Magnetic Resonance ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Hek293 Cells ◽  
Targeted Nanoparticles ◽  
Scanning Confocal Microscopy

Abstract Background The motif RXDLXXL-based nanoprobes allow specific imaging of integrin αvβ6, a protein overexpressed during tumorigenesis and tumor progression of various tumors. We applied a novel RXDLXXL-coupled cyclic arginine-glycine-aspartate (RGD) nonapeptide conjugated with ultrasmall superparamagnetic iron oxide nanoparticles (referred to as cFK-9-USPIO) for the application of integrin αvβ6-targeted magnetic resonance (MR) molecular imaging for breast cancer. Methods A novel MR-targeted nanoprobe, cFK-9-USPIO, was synthesized by conjugating integrin αvβ6-targeted peptide cFK-9 to N-amino (−NH2)-modified USPIO nanoparticles via a dehydration esterification reaction. Integrin αvβ6-positive mouse breast cancer (4 T1) and integrin αvβ6 negative human embryonic kidney 293 (HEK293) cell lines were incubated with cFK-9-AbFlour 647 (blocking group) or cFK-9-USPIO (experimental group), and subsequently imaged using laser scanning confocal microscopy (LSCM) and 3.0 Tesla magnetic resonance imaging (MRI) system. The affinity of cFK-9 targeting αvβ6 was analyzed by calculating the mean fluorescent intensity in cells, and the nanoparticle targeting effect was measured by the reduction of T2 values in an in vitro MRI. The in vivo MRI capability of cFK-9-USPIO was investigated in 4 T1 xenograft mouse models. Binding of the targeted nanoparticles to αvβ6-positive 4 T1 tumors was determined by ex vivo histopathology. Results In vitro laser scanning confocal microscopy (LSCM) imaging showed that the difference in fluorescence intensity between the targeting and blocking groups of 4 T1 cells was significantly greater than that in HEK293 cells (P < 0.05). The in vitro MRI demonstrated a more remarkable T2 reduction in 4 T1 cells than in HEK293 cells (P < 0.001). The in vivo MRI of 4 T1 xenograft tumor-bearing nude mice showed significant T2 reduction in tumors compared to controls. Prussian blue staining further confirmed that αvβ6 integrin-targeted nanoparticles were specifically accumulated in 4 T1 tumors and notably fewer nanoparticles were detected in 4 T1 tumors of mice injected with control USPIO and HEK293 tumors of mice administered cFK-9-USPIO. Conclusions Integrin αvβ6-targeted nanoparticles have great potential for use in the detection of αvβ6-overexpressed breast cancer with MR molecular imaging.

scholarly journals LncRNA APTR Promotes Uterine Leiomyoma Cell Proliferation by Targeting ERα to Activate the Wnt/β-Catenin Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Weiqiang Zhou ◽  
Guocheng Wang ◽  
Bilan Li ◽  
Junjie Qu ◽  
Yongli Zhang
Keyword(s):  
Cell Proliferation ◽  
Noncoding Rna ◽  
Uterine Leiomyoma ◽  
Laser Scanning ◽  
Wnt Pathway ◽  
Molecular Mechanisms ◽  
Wnt Signaling Pathway ◽  
Laser Scanning Confocal Microscopy

The molecular mechanisms by which uterine leiomyoma (UL) cells proliferate are unclear. Long noncoding RNA (lncRNA) is reported to participate in the occurrence and development of gynecological cancers. We investigated the molecular mechanisms that lncRNA uses in UL. We found that lncRNA Alu-mediated p21 transcriptional regulator (APTR) showed higher expression in UL tumor tissues compared with that in normal uterine tissues. APTR induced cell proliferation and colony formation both in vitro and in vivo. The JASPAR database showed that APTR was likely interacted with ERα, and these molecules were identified via laser scanning confocal microscopy and RNA immunoprecipitation analysis. To verify the correlation between APTR and ERα, we overexpressed and underexpressed APTR and simultaneously expressed ERα. The results showed that APTR function was suppressed. APTR increased the expressions of the proteins in the Wnt pathway, and inhibiting ERα eliminated these responses. In conclusion, our data suggest that APTR promoted leiomyoma cell proliferation through the Wnt pathway by targeting ERα, suggesting a new role of APTR in the Wnt signaling pathway in UL.

scholarly journals HDAC inhibitors as antifibrotic drugs in cardiac and pulmonary fibrosis

2019 ◽  
Vol 10 ◽  
pp. 204062231986269 ◽  
Author(s):  
Xing Lyu ◽  
Min Hu ◽  
Jieting Peng ◽  
Xiangyu Zhang ◽  
Yan Y Sanders
Keyword(s):  
Pulmonary Fibrosis ◽  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Wound Repair ◽  
Hdac Inhibitors ◽  
Scar Tissue ◽  
Nonhistone Proteins ◽  
Fibrotic Diseases

Fibrosis usually results from dysregulated wound repair and is characterized by excessive scar tissue. It is a complex process with unclear mechanisms. Accumulating evidence indicates that epigenetic alterations, including histone acetylation, play a pivotal role in this process. Histone acetylation is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are enzymes that remove the acetyl groups from both histone and nonhistone proteins. Aberrant HDAC activities are observed in fibrotic diseases, including cardiac and pulmonary fibrosis. HDAC inhibitors (HDACIs) are molecules that block HDAC functions. HDACIs have been studied extensively in a variety of tumors. Currently, there are four HDACIs approved by the US Food and Drug Administration for cancer treatment yet none for fibrotic diseases. Emerging evidence from in vitro and in vivo preclinical studies has presented beneficial effects of HDACIs in preventing or reversing fibrogenesis. In this review, we summarize the latest findings of the roles of HDACs in the pathogenesis of cardiac and pulmonary fibrosis and highlight the potential applications of HDACIs in these two fibrotic diseases.

Morphological assessment of the development of multinucleated giant cells in glioma by using mitosis-specific phosphorylated antibodies

2003 ◽  
Vol 98 (4) ◽  
pp. 854-859 ◽  
Author(s):  
Kenkou Maeda ◽  
Masaaki Mizuno ◽  
Toshihiko Wakabayashi ◽  
Syuntarou Takasu ◽  
Tetsurou Nagasaka ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Giant Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Multinucleated Giant Cells ◽  
Content Type ◽  
Scanning Confocal Microscopy ◽  
Drug Treatments ◽  
Fine Print

Object. The nature and origin of multinucleated giant cells in glioma have not been made clear. To investigate the phosphorylation of intermediate filaments, the authors studied multinucleated giant cells in vitro and in vivo by using mitosis-specific phosphorylated antibodies. Methods. Cultured human glioma cells were immunostained with monoclonal antibodies (mAbs) 4A4, KT13, and TM71, which recognized the phosphorylation of vimentin at Ser55, glial fibrillary acidic protein at Ser13, and vimentin at Ser71, respectively. Subsequently, the nature of multinucleated giant cells was investigated using laser scanning confocal microscopy. In addition, paraffin-embedded tissue sections obtained in three patients with giant cell glioblastoma were also investigated. Multinucleated giant cells were immunoreacted with the mAb 4A4 and not with KT13 and TM71 in vitro and in vivo. In addition, the authors obtained these results in multinucleated giant cells under natural conditions, without drug treatments. Conclusions. Findings in this investigation indicated that multinucleated giant cells are those remaining in mitosis between metaphase and telophase, undergoing neither fusion nor degeneration.

Integrin α2β1 (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1804-1809 ◽  
Author(s):  
Joachim Werr ◽  
Joakim Johansson ◽  
Einar E. Eriksson ◽  
Per Hedqvist ◽  
Erkki Ruoslahti ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Critical Role ◽  
Collagen Gel ◽  
Time Lapse ◽  
Surface Expression ◽  
Laser Scanning Confocal Microscopy ◽  
Rat Mesentery

Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the 2β1 (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the 2β1 integrin reduced PMN migration velocity in the extravascular tissue by 73% ± 3% and 70% ± 10%, respectively ( means ± SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-2 mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the 4β1 or 5β1integrins. Further evidence for a critical role of expression of 2β1 integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-2 mAb treatment. Thus, expression of 2β1 integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated.

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