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PubMed2006

High-purity selection and maintenance of gene expression in human neuroblastoma cells stably over-expressing GFP fusion protein. Application for opioid receptors desensitization studies.

Chronic use of opiates such as morphine is associated with drug tolerance, which is correlated with the desensitization of opioid receptors. This latter process involves phosphorylation of opioid receptors by G protein-coupled receptors kinases (GRKs) and subsequent uncoupling by beta-arrestins. To explore these molecular mechanisms, neuronal cell lines, endogenously expressing the opioid receptors, provide an ideal cellular model. Unfortunately, there are two major drawbacks: (1) these cells are refractory to cDNA introduction, resulting in low transfection efficiency; (2) continuous culturing of transfected cells invariably leads to phenotypic drift of the cultures even after an antibiotic selection. So, these cells were dropped in favor of heterologous expression systems, which are easier to transfect but whose relevance as adequate cellular model for studying opioid receptor regulation should be questioned, as recently demonstrated by [Haberstock-Debic, H., Kim, K.A.,Yu, Y.J., von Zastrow, M., 2005. Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J. Neurosci. 25, 7847-7857]. In this work, we describe a method, based on fluorescence-activated cell sorting (FACS), to select and maintain a high proportion of transfected SK-N-BE cells (a neuronal cell line endogenously expressing human Delta-Opioid Receptor (hDOR)), expressing the beta-arrestin1 fused to green fluorescent protein (GFP). While in functional experiments, we were not able to observe a major effect in non-sorted SK-N-BE cells expressing beta-arrestin1-GFP, the enrichment by 18-fold with FACS resulted in a robust increase of beta-arrestin1-GFP expression associated with strong hDOR desensitization. Moreover, this method also allows to counteract the phenotypic drift and to maintain a high-purity selection of SK-N-BE cells expressing beta-arrestin1-GFP. Thus, this approach provides a valuable tool for exploring opioid receptors desensitization in neuronal cells.

Analysis of Variance↗

PubMed2005

Time courses of B7 family molecules expressed on activated T-cells and their biological significance.

B7 family molecules are mainly expressed on the outer membrane of antigen-presenting cells. Here, our results demonstrate that CD80, CD86, and PD-L1 molecules are also expressed on T-cells that have been activated by simultaneous exposure to anti-CD3 and anti-CD28 mAbs, but PD-L2 and GL50 molecules were not detectable during the first six days of culture that follow such stimulation. We have analysed the time course of B7 family molecule expression on activated T-cells. CD28 and its ligands, CD80/CD86, have a high degree of co-localization and exhibit compartmental distribution on the membrane of activated T-cells, which is visualized by confocal microscopy. Interestingly, the co-localization of PD-1 and its ligand also exhibit similar phenomenon. Additionally, we provide evidence indicating that the CD80, CD86, and PD-L1 molecules are functional, since T-cells expressing B7 family molecules are able to stimulate the proliferation of highly purified allogeneic or autologous T-cells. Anti-CD80, anti-CD86, and soluble CD28-Ig protein could significantly attenuate the proliferation of T-cells, whereas anti-PD-L1 mAb may lead to the expansion of activated T-cells. We can conclude that activated T-cells expressing B7 family molecules could act as "APC" to trigger purified T-cells, and B7 family molecules play important roles during the activation of T-cells. These results indicate a need for further work, exploring the regulatory roles these molecules may play in immune responses.

Animals↗

PubMed2006

In vivo imaging with cellular resolution of bone marrow cells transplanted into the ischemic brain of a mouse.

The aim of the study was to monitor in vivo and noninvasively the fate of single bone marrow cells (BMCs) transplanted into the ischemic brain of unirradiated mice. In vivo imaging was performed through a closed cranial window, throughout the 2 weeks following cell transplantation, using laser-scanning confocal fluorescence microscopy. The window was chronically implanted above the left parieto-occipital cortex in C57BL/6J adult mice. BMC (3 x 10(5) nucleated cells in 0.5 microL medium) from 5-week-old transgenic mice, ubiquitously expressing green fluorescent protein (GFP), was transplanted into the ipsilateral cortex 24 h after the induction of focal ischemia by coagulation of the left middle cerebral artery (n = 15). Three nonischemic mice served as controls. Repeated in vivo imaging, up to a depth of 200 microm, revealed that BMCs survived within the ischemic and peri-ischemic cortex, migrated significantly towards the lesion, proliferated and adopted a microglia-like morphology over 2 weeks. These results were confirmed using ex vivo imaging after appropriate immunocytochemical treatments. This study indicates that confocal fluorescence microscopy is a reliable and unique tool to repeatedly assess with cellular resolution the in vivo dynamic fate of fluorescent cells transplanted into a mouse brain. These results also provide the first in vivo findings on the fate of single BMCs transplanted into the ischemic brain of unirradiated mice.

Animals↗

PubMed2002

Two C-terminal cysteines are necessary for proper folding of the peptidase neprilysin/CD10.

Neprilysin (NEP) consists of 749 amino acids with two conserved cysteines (734, 746) and a putative CAAX motif (residues 746-749, CRVW) at the C-terminus. To investigate the role of the C-terminal conserved cysteine residues, three NEP mutants (C734S, C746S, and double mutant C734S/C746S) were constructed by use of site-directed mutagenesis. Western blot analysis of lysates of transfected cells revealed the presence of three NEP forms in wild type and mutants with a different glycosylation pattern. Point mutations of C734 as well as C746 by serine dramatically diminished the plasma membrane association of NEP as detected by flow cytometry and laser scanning microscopy. Endoprotease enzyme activity was slightly diminished in the C746S-NEP variant and was not detectable in the C734S-form of NEP suggesting a pivotal role of the C734 in the proper folding of the enzyme. Prenylation of NEP was not detected in an in vivo assay.

Base Sequence↗

PubMed2005

IKK mediates ischemia-induced neuronal death.

The IkappaB kinase complex IKK is a central component of the signaling cascade that controls NF-kappaB-dependent gene transcription. So far, its function in the brain is largely unknown. Here, we show that IKK is activated in a mouse model of stroke. To investigate the function of IKK in brain ischemia we generated mice that contain a targeted deletion of Ikbkb (which encodes IKK2) in mouse neurons and mice that express a dominant inhibitor of IKK in neurons. In both lines, inhibition of IKK activity markedly reduced infarct size. In contrast, constitutive activation of IKK2 enlarged the infarct size. A selective small-molecule inhibitor of IKK mimicked the effect of genetic IKK inhibition in neurons, reducing the infarct volume and cell death in a therapeutic time window of 4.5 h. These data indicate a key function of IKK in ischemic brain damage and suggest a potential role for IKK inhibitors in stroke therapy.

Animals↗

PubMed2006

Fluorescence cross-correlation spectroscopy in living cells.

Cell biologists strive to characterize molecular interactions directly in the intracellular environment. The intrinsic resolution of optical microscopy, however, allows visualization of only coarse subcellular localization. By extracting information from molecular dynamics, fluorescence cross-correlation spectroscopy (FCCS) grants access to processes on a molecular scale, such as diffusion, binding, enzymatic reactions and codiffusion, and has become a valuable tool for studies in living cells. Here we review basic principles of FCCS and focus on seminal applications, including examples of intracellular signaling and trafficking. We consider FCCS in the context of fluorescence resonance energy transfer and multicolor imaging techniques and discuss application strategies and recent technical advances.

Algorithms↗

PubMed2004

Role of nuclear factor-kappaB and heme oxygenase-1 in the mechanism of action of an anti-inflammatory chalcone derivative in RAW 264.7 cells.

The synthetic chalcone 3',4',5',3,4,5-hexamethoxy-chalcone (CH) is an anti-inflammatory compound able to reduce nitric oxide (NO) production by inhibition of inducible NO synthase protein synthesis. In this work, we have studied the mechanisms of action of this compound. CH (10-30 microm) prevents the overproduction of NO in RAW 264.7 macrophages stimulated with lipopolysaccharide (1 microg ml(-1)) due to the inhibition of nuclear factor kappaB (NF-kappaB) activation. We have shown that treatment of cells with CH results in diminished degradation of the NF-kappaB-IkappaB complex leading to inhibition of NF-kappaB translocation into the nucleus, DNA binding and transcriptional activity. We also demonstrate the ability of this compound to activate NfE2-related factor (Nrf2) and induce heme oxygenase-1 (HO-1). Our results indicate that CH determines a rapid but nontoxic increase of intracellular oxidative species, which could be responsible for Nrf2 activation and HO-1 induction by this chalcone derivative. This novel anti-inflammatory agent simultaneously induces a cytoprotective response (HO-1) and downregulates an inflammatory pathway (NF-kappaB) with a mechanism of action different from antioxidant chalcones.

Animals↗

PubMed2004

Centrosome amplification induced by DNA damage occurs during a prolonged G2 phase and involves ATM.

Centrosomes are the principal microtubule organising centres in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, we examined cells that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrested in G2 phase and formed supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurred without an additional DNA replication round and was not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduced DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification was potentiated by Rad54 disruption. Gene targeting of ATM reduced, but did not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. Our data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints.

Androstadienes↗

PubMed2006

Quantitative analysis of melanocytic tissue array reveals inverse correlation between activator protein-2alpha and protease-activated receptor-1 expression during melanoma progression.

The identification of molecular markers of melanoma progression is needed to more accurately stage and identify treatments for patients with malignant melanoma. Previously, we demonstrated that loss of the activator protein-2alpha (AP-2alpha) expression results in overexpression of the protease-activated receptor-1 (PAR-1) in human melanoma cell lines. Here, we used a tissue microarray platform that consisted of 64 melanocytic lesions, including dysplastic nevi (N=21), primary melanoma (N=20), and metastatic melanoma (N=23). We analyzed the expression of AP-2 and PAR-1 simultaneously by immunofluorescent microscopy with an automated quantification laser scanning cytometer. AP-2 was highly expressed in normal cutaneous melanocytes and dysplastic nevi but not in melanoma metastases. We observed a significantly higher number of AP-2-positive cells in the dysplastic nevi (P=0.0013) and primary melanoma (P=0.0023) compared to the metastatic melanoma. In contrast, we observed a significantly higher percentage of PAR-1-positive cells in the metastatic melanoma compared to dysplastic nevi (P=0.0072) and primary melanoma (P=0.0138). Increased expression of PAR-1 in metastatic melanomas contributes to tumor progression by modulating expression of genes, such as IL-8, matrix metalloproteinase-2, vascular endothelial growth factor, platelet-derived growth factor, and integrins. These findings support our hypothesis that loss of AP-2 is a crucial event in the progression of human melanoma and contributes to the acquisition of the metastatic phenotype via upregulation of PAR-1.

Disease Progression↗

PubMed2006

Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging.

Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin-Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein-tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting-contrary to expectations-that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.

Animals↗

PubMed2006

Controversy fuels trafficking of GPI-anchored proteins.

The model that glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) take a direct transport route to the apical membrane of epithelial cells has recently been challenged. In this issue, Paladino et al. (p. 1023) and Hua et al. (p. 1035) show that the original view nevertheless holds. This closes a chapter in the winding story of GPI-AP trafficking but opens another phase, as the controversy has stimulated the development of new methodology.

Animals↗

PubMed2006

Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement.

Cells lining the gastrointestinal tract serve as both a barrier to and a pathway for infectious agent entry. Dendritic cells (DCs) present in the lamina propria under the columnar villus epithelium of the small bowel extend processes across this epithelium and capture bacteria, but previous studies provided limited information on the nature of the stimuli, receptors, and signaling events involved in promoting this phenomenon. Here, we use immunohistochemical as well as dynamic explant and intravital two-photon imaging to investigate this issue. Analysis of CD11c-enhanced green fluorescent protein (EGFP) or major histocompatibility complex CII-EGFP mice revealed that the number of trans-epithelial DC extensions, many with an unusual "balloon" shape, varies along the length of the small bowel. High numbers of such extensions were found in the proximal jejunum, but only a few were present in the terminal ileum. The extensions in the terminal ileum markedly increased upon the introduction of invasive or noninvasive Salmonella organisms, and chimeric mouse studies revealed the key role of MyD88-dependent Toll-like receptor (TLR) signaling by nonhematopoietic (epithelial) elements in the DC extension response. Collectively, these findings support a model in which epithelial cell TLR signaling upon exposure to microbial stimuli induces active DC sampling of the gut lumen at sites distant from organized lymphoid tissues.

Animals↗

PubMed2004

The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles.

We report the characterization of Cep170, a forkhead-associated (FHA) domain protein of previously unknown function. Cep170 was identified in a yeast two-hybrid screen for interactors of Polo-like kinase 1 (Plk1). In human cells, Cep170 is constantly expressed throughout the cell cycle but phosphorylated during mitosis. It interacts with Plk1 in vivo and can be phosphorylated by Plk1 in vitro, suggesting that it is a physiological substrate of this kinase. Both overexpression and small interfering RNA (siRNA)-mediated depletion studies suggest a role for Cep170 in microtuble organization and cell morphology. Cep170 associates with centrosomes during interphase and with spindle microtubules during mitosis. As shown by immunoelectron microscopy, Cep170 associates with subdistal appendages, typical of the mature mother centriole. Thus, anti-Cep170 antibodies stain only one centriole during G1, S, and early G2, but two centrioles during late G2 phase of the cell cycle. We show that Cep170 labeling can be used to discriminate bona fide centriole overduplication from centriole amplification that results from aborted cell division.

Alternative Splicing↗

PubMed2006

Tolfenamic acid and pancreatic cancer growth, angiogenesis, and Sp protein degradation.

BACKGROUND: Sp1, Sp3, and Sp4 are transcription factors that regulate cell proliferation and vascular endothelial growth factor (VEGF) expression and are overexpressed in many cancer cell lines. For some cancers, Sp1 overexpression is associated with poor survival. Cyclooxygenase inhibitors decrease Sp1 expression in cancer cells, and therefore different structural classes of nonsteroidal anti-inflammatory drugs (NSAIDs) were screened for their ability to decrease levels of Sp1, Sp3, and Sp4 and to decrease pancreatic tumor growth and metastasis in an in vivo model. METHODS: Levels of Sp1, Sp3, Sp4, and VEGF proteins in pancreatic cancer cell lines were assessed by immunoblot analysis. mRNA was assessed by reverse transcription-polymerase chain reaction. Panc-1 pancreatic cancer cells transfected with VEGF promoter constructs were used to assess VEGF promoter activation. Pancreatic tumor weight and size and liver metastasis were assessed in an orthotopic mouse model of pancreatic cancer (groups of 10 mice). Protein expression in tumors was assessed immunohistochemically. RESULTS: Tolfenamic acid and structurally related biaryl derivatives induced degradation of Sp1, Sp3, and Sp4 in pancreatic cancer cells. Tolfenamic acid also inhibited VEGF mRNA and protein expression in pancreatic cancer cells; this inhibition was associated with the decreased Sp-dependent activation of the VEGF promoter. In the mouse model for pancreatic cancer, treatment with tolfenamic acid (50 mg/kg of body weight), compared with control treatment, statistically significantly decreased tumor growth and weight (P = .005), liver metastasis (P = .027), and levels of Sp3 and VEGF (P = .009) and Sp1 and Sp4 (P = .006) proteins in tumors. For example, tumors from mice treated with tolfenamic acid (50 mg/kg) had statistically significantly lower VEGF levels (45%, 95% confidence interval = 39% to 51%; P = .009) than tumors from control mice. CONCLUSIONS: Tolfenamic acid is a new antipancreatic cancer NSAID that activates degradation of transcription factors Sp1, Sp3, and Sp4; reduces VEGF expression; and decreases tumor growth and metastasis.

Animals↗

PubMed2006

Sinking velocities of phytoplankton measured on a stable density gradient by laser scanning.

Two particular difficulties in measuring the sinking velocities of phytoplankton cells are preventing convection within the sedimenting medium and determining the changing depth of the cells. These problems are overcome by using a density-stabilized sedimentation column scanned by a laser. For freshwater species, a suspension of phytoplankton is layered over a vertical density gradient of Percoll solution; as the cells sink down the column their relative concentration is measured by the forward scattering of light from a laser beam that repeatedly scans up and down the column. The Percoll gradient stabilizes the column, preventing vertical mixing by convection, radiation or perturbation of density by the descending cells. Measurements were made on suspensions of 15 microm polystyrene microspheres with a density of 1050 kg m-3; the mean velocity was 6.28 microm s-1, within 1.5% of that calculated by the Stokes equation, 6.36 microms-1. Measurements made on the filamentous cyanobacterium Planktothrix rubescens gave mean velocities within the theoretical range of values based on the range of size, shape, orientation and density of the particles in a modified Stokes equation. Measurements on marine phytoplankton may require density gradients prepared with other substances.

Cyanobacteria↗

PubMed2006

Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro.

We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival keratinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type 16 (HPV16). The approach adopted is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interactions via diffusible growth factors. Generally, IHGK reveal transcription of the HPV16 E6/E7 gene at rising passages. Fluorescence in situ hybridization performed for chromosomes 1, 8, 10, and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant. Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors interleukin 1beta, granulocyte-macrophage colony stimulating factor, fibroblast growth factor 7, and EGF in OCCs is different from that in MCs. When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant growth. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro.

Animals↗

PubMed2005

Retinal nerve fiber layer thickness is unaffected in migraine patients.

OBJECTIVES: To search for possible structural effects of migraine on the retina. MATERIALS AND METHODS: The study group included 39 consecutive migraine patients, 15 with visual aura and 24 without aura, and 25 healthy subjects as the control group. Scanning laser polarimetry was used to measure and compare retinal nerve fiber layer (RNFL) thickness between migraine and control groups. RESULTS: The mean age of the patients and the control group were 35.64 +/- 8.3 and 35.96 +/- 9.14 years, respectively (P = 0.92) and the mean duration of migraine in patients with aura and without aura as 4.4 +/- 2.9 and 5.3 +/- 4.3 years, respectively (P = 0.68). Parameters related to RNFL thickness were found to be similar (P > 0.001) in migraine and control subjects. After examining the whole patient group migraine patients with and without aura were compared to each other and the control group individually for RNFL thickness parameters. Briefly no statistical difference was found for any of the test parameters between migraine patients with aura, without aura and controls (P > 0.001). CONCLUSION: Retinal nerve fiber layer thickness was found to be unaffected in migraine patients.

Adolescent↗

PubMed2006

Method for assessment of viability and morphological changes of bacteria in the early stage of colony formation on a simulated natural environment.

A quantitative analysis of changes in the physiological status of bacterial cells is a fundamental type of study in microbiological research. We devised a method for measuring the viability of bacteria in the early stage of colony formation on a simulated natural environment. In this method, a solid medium containing soil extract was used, and the formation of bacterial microcolonies on a membrane filter was determined by use of a laser scanning cytometer combined with live-dead fluorescent dyes. A polychlorinated biphenyl degrader, Comamonas testosteroni TK102, was used in this study. Surprisingly, approximately 20% of the microcolonies had their growth stopped and eventually died. In the presence of biphenyl, the growth arrest was increased to 50%, and filamentous cells were observed in the colonies. Predicted intermediate metabolites of biphenyl were added to the medium to determine the relationship between the change of viability and the production of metabolites, and the addition of 2,3-dihydroxybiphenyl showed low viability. The arrest was not observed to occur on nutrient-rich medium, suggesting that the change in viability might occur in a nutrient-poor natural condition. The results of this study demonstrated that toxic metabolites of xenobiotics might change cell viability in the natural environment.

Bacteria↗