Phosphodiesterase 5 Inhibitor Potentiates Epigallocatechin 3-O-Gallate-Induced Apoptotic Cell Death via Activation of the cGMP Signaling Pathway in Caco-2 Cells

Epigallocatechin 3-O-gallate (EGCG) is a predominant component in green tea with various health benefits. The 67 kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that is overexpressed in various types of cancer; 67LR was identified a cell surface EGCG target that plays a pivotal role in tumor growth, metastasis, and resistance to chemotherapy. However, the plasma concentration of EGCG is limited, and its molecular mechanisms remain unelucidated in colon cancer. In this study, we found that the phosphodiesterase 5 (PDE5) inhibitor, vardenafil (VDN), potentiates EGCG-induced apoptotic cell death in colon cancer cells. The combination of EGCG and VDN induced apoptosis via activation of the endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase Cδ signaling pathway. In conclusion, the PDE5 inhibitor, VDN, may reduce the intracellular PDE5 enzyme activity that potentiates EGCG-induced apoptotic cell death in Caco-2 cells. These results suggest that PDE5 inhibitors can be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death. Therefore, EGCG may be employed as a therapeutic candidate for colon cancer.     

A dual fluorescent reporter for the investigation of methionine mistranslation in live cells

In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNA^Glu mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRNA^Glu restores fluorescence in proportion to the amount of misacylated tRNA^Glu. This cellular approach works well for both transient transfection and established stable HEK293 lines. It is rapid, straightforward, and well suited for high-throughput activity analysis under a wide range of physiological conditions. As a proof of concept, we apply this method to characterize the effect of human tRNA^Glu isodecoders on mistranslation and discuss the implications of our findings.     

Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice

Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B‐CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε‐globin‐histone H2B‐CFP line for multicolor imaging and flow cytometry analysis. The ε‐globin‐H2B‐CFP line was used to analyze the cell cycle distribution and proliferation of CFP‐expressing primitive erythroblasts from E8.5‐E13.5. We also evaluated phagocytosis of extruded CFP‐positive nuclei by macrophages in fetal liver and placenta. The ε‐globin‐H2B‐CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage. genesis 51:751–762. © 2013 Wiley Periodicals, Inc.     

A fluorescent reporter for mapping cellular protein‐protein interactions in time and space

We introduce a fluorescent reporter for monitoring protein–protein interactions in living cells. The method is based on the Split‐Ubiquitin method and uses the ratio of two auto‐fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two‐channel time‐lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio‐temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran‐GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein–protein interactions identified from large‐scale screens can be effectively followed up by high‐resolution single‐cell analysis.     

The IRG mouse: a two-color fluorescent reporter for assessing Cre-mediated recombination and imaging complex cellular relationships in situ

The Cre-loxP system is widely used for making conditional alterations to the mouse genome. Cre-mediated recombination is frequently monitored using reporter lines in which Cre expression activates a reporter gene driven by a ubiquitous promoter. Given the distinct advantages of fluorescent reporters, we developed a transgenic reporter line, termed IRG, in which DsRed-Express, a red fluorescent protein (RFP) is expressed ubiquitously prior to Cre-mediated recombination and an enhanced green fluorescent protein (EGFP) following recombination. Besides their utility for monitoring Cre-mediated recombination, we show that in IRG mice red and green native fluorescence can be imaged simultaneously in thick tissue sections by confocal microscopy allowing for complex reconstructions to be created that are suitable for analysis of neuronal morphologies as well as neurovascular interactions in brain. IRG mice should provide a versatile tool for analyzing complex cellular relationships in both neural and nonneural tissues.     

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.     

Reexamination of Opioid Stimulation of cGMP Formation in Cell Lines of Neuronal Origin

1. The present study reexamines a previous notion on opioid stimulation of cyclic GMP (cGMP) formation and the retraction of the original findings.2. The effect of opioid agonists on cGMP accumulation in two cell lines of neuronal origin was measured. The proportion of cGMP stimulation in NG108-15 neuroblastoma × glioma hybrid cells resembled the proportion of [Ca²⁺]_in elevation by opioids in this culture. The failure of opioids to stimulate cGMP formation in SK-N-SH human neuroblastoma coincided with the lack of cGMP stimulation by other Ca²⁺ mobilizing agents in these cells. The nitric oxide donor nitroprusside elevated cGMP in both cell lines.3. The implication of the opioid-Ca²⁺-NO-cGMP cellular pathway for opioid activity in vivo is discussed.     

Measurement of the intracellular pH of blood platelets using a photolabel fluorescent probe

The internal pH of blood platelets using the intracellular photolabel probe azidofluorescein diacetate was determined. No change of intracellular pH during thrombin activation of human platelets was observed. Platelets were found to adjust themselves very quickly to the external pH. Quantitative subcellular localization of the attachment sites of this probe reveals that most of it is bound to low molecular weight proteins or peptides.     

Occurrence of adenosine 3′:5′-cyclic monophosphate in plant tissues

A procedure is described which unequivocally demonstrates the presence of adenosine 3′:5′-cyclic monophosphate in Phaseolus vulgaris. Its concentration was determined spectrophotometrically at 2·6–9·2 nmol g^?1 of tissue (dry wt) for 6-day-old seedlings and about one-tenth of this in 13-day-old plants.     

Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation

Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs’ physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, and Western Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes.     

Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells

The cellular prion protein (PrP(c)) is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein whose conformational altered forms (PrP(sc)) are known to cause neurodegenerative diseases in mammals. In order to investigate the intracellular traffic of mammalian PrP(c) in living cells, we have generated a green fluorescent protein (GFP) tagged version of PrP(c). The recombinant protein was properly anchored at the cell surface and its distribution pattern was similar to that of the endogenous PrP(c), with labeling at the plasma membrane and in an intracellular perinuclear compartment. Comparison of the steady-state distribution of GFP-PrP(c) and two N-terminal deletion mutants (Delta32-121 and Delta32-134), that cause neurological symptoms when expressed in PrP knockout mice, was carried out. The mutant proteins accumulated in the plasma membrane at the expense of decreased labeling in the perinuclear region when compared with GFP-PrP(c). In addition, GFP-PrP(c), but not the two mutants, internalized from the plasma membrane in response to Cu2+ treatment and accumulated at a perinuclear region in SN56 cells. Our data suggest that GFP-PrP(c) can be used to follow constitutive and induced PrP(c) traffic in living cells.     

Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes

This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at -80 degrees C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY-14) and propidium iodide.Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20-30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.     

Testicular somatic cells in the stony coral Euphyllia ancora express an endogenous green fluorescent protein

In a variety of organisms, adult gonads contain several specialized somatic cells that regulate and support the development of germline cells. In stony corals, the characteristics and functions of gonadal somatic cells remain largely unknown. No molecular markers are currently available that allow for the identification and enrichment of gonadal somatic cells in corals. Here, we showed that the testicular somatic cells of a stony coral, Euphyllia ancora, express an endogenous green fluorescent protein (GFP). Fluorescence microscopy showed that, in contrast to the endogenous expression of the red fluorescent protein of E. ancora ovaries that we have previously reported, the testes displayed a distinct green fluorescence. Molecular identification and spectrum characterization demonstrated that E. ancora testes expressed a GFP (named EaGFP) that is a homolog of the GFP from the jellyfish Aequorea victoria and that possesses an excitation maximum of 506 nm and an emission maximum of 514 nm. Immunohistochemical analyses revealed that the testicular somatic cells, but not the germ cells, expressed EaGFP. EaGFP was enclosed within one or a few granules in the cytoplasm of testicular somatic cells, and the granule number decreased as spermatogenesis proceeded. We also showed that testicular somatic cells could be enriched by using endogenous GFP as an indicator. The present study not only revealed one of the unique cellular characteristics of coral testicular cells but also established a technical basis for more in‐depth investigations of the function of testicular somatic cells in spermatogenesis in future studies.     

Multiple gene detection by in situ RT-PCR in isolated plant cells and tissues

With the number of functional genomic approaches in plant biology increasing daily, the demand for rapid and reliable RNA localization techniques for gene characterization is being felt. We present herein a novel, liquid phase in situ RT-PCR (IS-RT-PCR) protocol using a combination of gene-specific fluorescent primers and spectral confocal microscopy to localize target RNA in epicotyl sections and xylogenic suspension cultures of Zinnia elegans. Potential sources of artefacts from fixation to gene detection were systematically eliminated using both fluorescent primers and nucleotides for 18S rRNA gene detection, resulting in a set of optimal parameters for IS-RT-PCR that may be readily adapted to any target gene. By judiciously choosing fluorescent primers with non-overlapping fluorochromes, we have shown that our technique is readily adapted to multiplex IS-RT-PCR, enabling the simultaneous localization of more than one gene within a complex tissue or heterogeneous cell population. A 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (6-HEX)-labelled primer and a tetrachloro-6-carboxy-fluorescein (TET)-labelled primer were designed for two marker genes associated with programmed cell death in tracheary elements (TEs): an endonuclease (Zen1) and a cysteine protease (ZcP4), respectively. An additional Cyan5 (Cy5)-labelled primer was used to monitor 18SrRNA expression. As expected, the 18S signal was constitutively expressed throughout epicotyls sections and living cells in xylogenic in vitro cultures, whereas Zen1 and ZcP4 were co-localized in forming TEs both in planta and in vitro. Analogous to clustering analysis of gene expression using microarrays to elucidate common metabolic pathways and developmental processes, this novel technique is perfectly adapted to gaining a better understanding of gene function via the coordinated expression of genes in specific cell types of complex tissues and cell populations.     

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.     

Effect of zinc on cellular levels of calmodulin and cyclic adenosine monophosphate in the adipocyte

A perturbation of zinc metabolism has been noted in subjects with obesity. Zinc may also participate in the intracellular signal cascade by affecting cellular calcium influx and a change in the calcium-calmodulin (CaM)-cyclic adenosine monophosphate (cAMP) pathway. The possible effects of zinc on cellular concentrations of CaM, a major cytosolic calcium-binding protein, in the adipocytes derived from obese (ob/ob) mice and their lean counterparts were studied. Adipocytes derived from both phenotypes of mice were treated either with 0.2 mM of zinc sulfate or without any additive for 1 h of incubation; the cellular levels of CaM and cAMP were then determined. The results showed that the obese mice had lower CaM and cAMP levels in their adipocytes compared to the lean mice. Zinc treatment reduced CaM and increased cAMP levels in all mice, although this effect was more pronounced in the lean mice. This study indicated that there was an inverse interaction between CaM and cAMP in their cellular levels in the mouse adipocytes and that might be affected by exogenous zinc addition.     

Detecting oxygen consumption in the proximity of Saccharomyces cerevisiae cells using self-assembled fluorescent nanosensors

We describe a strategy for the preparation and self-assembly of fluorescent nanosensors onto Saccharomyces cerevisiae cell surfaces for dynamically measuring oxygen concentration in the proximity of living cells. Amine functionalized polystyrene nanobeads were impregnated with an oxygen-sensitive ruthenium(II) complex and the beads' surface was coated with polyethylenimine. The resulting nanosensors were assembled on individual S. cerevisiae cells in a controlled manner at physiological pH for continuously monitoring oxygen consumption. This approach exemplifies a general scheme for assembling fluorescent nanosensors on cells for the non-invasive, reversible, and real-time measurement of other physiologically relevant processes, such as the efflux of protons and carbon dioxide, or the influx of glucose.     

Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP

Physiologically, nitric oxide (NO) signal transduction occurs through soluble guanylyl cyclase (sGC), which catalyses cyclic GMP (cGMP) formation. Knowledge of the kinetics of NO-evoked cGMP signals is therefore critical for understanding how NO signals are decoded. Studies on cerebellar astrocytes showed that sGC undergoes a desensitizing profile of activity, which, in league with phosphodiesterases (PDEs), was hypothesized to diversify cGMP responses in different cells. The hypothesis was tested by examining the kinetics of cGMP in rat striatal cells, in which cGMP accumulated in neurones in response to NO. Based on the effects of selective PDE inhibitors, cGMP hydrolysis following exposure to NO was attributed to a cGMP-stimulated PDE (PDE 2). Analysis of NO-induced cGMP accumulation in the presence of a PDE inhibitor indicated that sGC underwent marked desensitization. However, the desensitization kinetics determined under these conditions described poorly the cGMP profile observed in the absence of the PDE inhibitor. An explanation shown plausible theoretically was that cGMP determines the level of sGC desensitization. In support, tests in cerebellar astrocytes indicated an inverse relationship between cGMP level and recovery of sGC from its desensitized state. We suggest that the degree of sGC desensitization is related to the cGMP concentration and that this effect is not mediated by (de)phosphorylation.     

检测大肠杆菌内c-di-GMP水平的双荧光素报告质粒的构建及应用

细菌通过调控第二信使环二鸟苷酸(cyclic diguanylate, c-di-GMP)而促进其适应环境、存活及致病。【目的】本研究旨在建立有效的c-di-GMP水平检测方法,为大肠杆菌内c-di-GMP水平检测提供便利条件。【方法】根据c-di-GMP核糖开关受体的调控方式、荧光报告基因等设计引物,通过重叠聚合酶链反应(overlap polymerase chain reaction, overlap PCR)和同源重组酶构成基于核糖开关的双荧光素报告质粒pAmCherry-Vc2EGFP(pACVcE),然后构建c-di-GMP代谢基因过表达菌株和缺失菌株,利用pACVcE检测大肠杆菌内c-di-GMP水平。【结果】OverlapPCR扩增产物与目的靶序列一致,测序结果证明pACVcE序列正确。表达c-di-GMP合成酶DgcZ的大肠杆菌胞内c-di-GMP水平显著升高,而表达c-di-GMP降解酶PdeK的大肠杆菌胞内c-di-GMP水平显著降低。禽致病性大肠杆菌的胞内c-di-GMP水平检测发现c-di-GMP降解酶基因pdeK缺失后胞内的c-di-GMP水平显著升高。【结...