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2.
Methods Mol Biol ; 1888: 1-20, 2019.
Article in English | MEDLINE | ID: mdl-30519938

ABSTRACT

PREDECT, a European IMI consortium, has assumed the task to generate robust 2D and 3D culture platforms. Protocols established for 2D and 3D monoculture and stromal coculture models of increasing complexity (spheroid, stirred-tank bioreactor, Matrigel- and collagen-embedded cultures) have been established between six laboratories within academia, biotech, and pharma. These models were tested using three tumor cell lines (MCF7, LNCaP, and NCI-H1437), covering three pathologies (breast, prostate, and lung), but should be readily transferable to other model systems. Fluorescent protein tagged cell lines were used for all platforms, allowing for online measurement of growth curves and drug responses to treatments. All methods, from culture setup to phenotypic characterization and gene expression profiling are described in this chapter.The adaptable methodologies and detailed protocols described here should help to include these models more readily to the drug discovery pipeline.


Subject(s)
Cell Culture Techniques , Bioreactors , Cell Line, Tumor , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression , Gene Order , Genes, Reporter , Genetic Vectors/genetics , Humans , Image Processing, Computer-Assisted , Microscopy, Fluorescence , Neoplasms/diagnosis , Neoplasms/drug therapy , Neoplasms/pathology , Software , Spheroids, Cellular , Transduction, Genetic , Tumor Cells, Cultured
3.
Stem Cell Reports ; 11(2): 363-379, 2018 08 14.
Article in English | MEDLINE | ID: mdl-30057263

ABSTRACT

Tauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

4.
Sci Data ; 4: 170170, 2017 11 21.
Article in English | MEDLINE | ID: mdl-29160867

ABSTRACT

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.


Subject(s)
Models, Biological , Neoplasms , Cell Culture Techniques , Humans , Imaging, Three-Dimensional
5.
EJNMMI Radiopharm Chem ; 1(1): 3, 2017.
Article in English | MEDLINE | ID: mdl-29564380

ABSTRACT

BACKGROUND: Phosphodiesterase 10A (PDE10A) is an important regulator of nigrostriatal dopamine (DA) neurotransmission. However, little is known on the effect of alterations in DA neurotransmission on PDE10A availability. Here, we used [18F]JNJ42259152 PET to measure changes in PDE10A availability, secondary to pharmacological alterations in DA release and to investigate whether these are D1- or D2-receptor driven. RESULTS: Acute treatment of rats using D-amphetamine (5 mg, s.c. and 1 mg/kg i.v.) did not result in a significant change in PDE10A BPND compared to baseline conditions. 5-day D-amphetamine treatment (5 mg/kg, s.c.) increased striatal PDE10A BPND compared to the baseline (+24 %, p = 0.03). Treatment with the selective D2 antagonist SCH23390 (1 mg/kg) and D-amphetamine decreased PDE10A binding (-22 %, p = 0.03). Treatment with only SCH23390 further decreased PDE10A binding (-26 %, p = 0.03). No significant alterations in PDE10A mRNA levels were observed. CONCLUSIONS: Repeated D-amphetamine treatment significantly increased PDE10A binding, which is not observed upon selective D1 receptor blocking. This study suggests a potential pharmacological interaction between PDE10A enzymes and drugs modifying DA neurotransmission. Therefore, PDE10A binding in patients with neuropsychiatric disorders might be modulated by chronic DA-related treatment.

6.
Sci Rep ; 6: 28951, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27364600

ABSTRACT

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.


Subject(s)
Coculture Techniques/methods , Spheroids, Cellular/cytology , Cell Line, Tumor , Humans , Imaging, Three-Dimensional , MCF-7 Cells , Stromal Cells/cytology
7.
PLoS One ; 11(6): e0156942, 2016.
Article in English | MEDLINE | ID: mdl-27303813

ABSTRACT

In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation.


Subject(s)
Cell Culture Techniques/methods , Imaging, Three-Dimensional/methods , Light , Microscopy, Confocal/methods , Spheroids, Cellular/cytology , Algorithms , Cancer-Associated Fibroblasts/cytology , Cell Line , Cell Line, Tumor , Cell Proliferation , Cell Survival , Computer Simulation , Humans , Image Processing, Computer-Assisted/methods , Models, Biological , Reproducibility of Results , Tumor Microenvironment
8.
Biotechnol J ; 9(9): 1115-28, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25174503

ABSTRACT

Cancers are complex and heterogeneous pathological "organs" in a dynamic interplay with their host. Models of human cancer in vitro, used in cancer biology and drug discovery, are generally highly reductionist. These cancer models do not incorporate complexity or heterogeneity. This raises the question as to whether the cancer models' biochemical circuitry (not their genome) represents, with sufficient fidelity, a tumor in situ. Around 95% of new anticancer drugs eventually fail in clinical trial, despite robust indications of activity in existing in vitro pre-clinical models. Innovative models are required that better capture tumor biology. An important feature of all tissues, and tumors, is that cells grow in three dimensions. Advances in generating and characterizing simple and complex (with added stromal components) three-dimensional in vitro models (3D models) are reviewed in this article. The application of stirred bioreactors to permit both scale-up/scale-down of these cancer models and, importantly, methods to permit controlled changes in environment (pH, nutrients, and oxygen) are also described. The challenges of generating thin tumor slices, their utility, and potential advantages and disadvantages are discussed. These in vitro/ex vivo models represent a distinct move to capture the realities of tumor biology in situ, but significant characterization work still remains to be done in order to show that their biochemical circuitry accurately reflects that of a tumor.


Subject(s)
Cell Culture Techniques/methods , In Vitro Techniques/methods , Neoplasms/pathology , Animals , Bioreactors , Cell Biology , Humans , Models, Biological
9.
PLoS One ; 9(1): e86892, 2014.
Article in English | MEDLINE | ID: mdl-24489801

ABSTRACT

Closure of the ductus arteriosus (DA) is a crucial step in the transition from fetal to postnatal life. Patent DA is one of the most common cardiovascular anomalies in children with significant clinical consequences especially in premature infants. We aimed to identify genes that specify the DA in the fetus and differentiate it from the aorta. Comparative microarray analysis of laser-captured microdissected endothelial (ECs) and vascular smooth muscle cells (SMCs) from the DA and aorta of fetal rats (embryonic day 18 and 21) identified vessel-specific transcriptional profiles. We found a strong age-dependency of gene expression. Among the genes that were upregulated in the DA the regulator of the G-protein coupled receptor 5 (Rgs5) and the transcription factor distal-less homeobox 1 (Dlx1) exhibited the highest and most significant level of differential expression. The aorta showed a significant preferential expression of the Purkinje cell protein 4 (Pcp4) gene. The results of the microarray analysis were validated by real-time quantitative PCR and immunohistochemistry. Our study confirms vessel-specific transcriptional profiles in ECs and SMCs of rat DA and aorta. Rgs5 and Dlx1 represent novel molecular targets for the regulation of DA maturation and closure.


Subject(s)
Ductus Arteriosus/metabolism , Endothelial Cells/metabolism , Gene Expression Profiling , Homeodomain Proteins/genetics , Laser Capture Microdissection , Myocytes, Smooth Muscle/metabolism , RGS Proteins/genetics , Transcription Factors/genetics , Animals , Female , Homeodomain Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Organ Specificity/genetics , RGS Proteins/metabolism , Rats, Wistar , Transcription Factors/metabolism , Transcription, Genetic
10.
J Biomol Screen ; 18(1): 54-66, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22923784

ABSTRACT

For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , High-Throughput Screening Assays/methods , Tumor Microenvironment , Cell Culture Techniques , Cell Line, Tumor , Coculture Techniques , Drug Screening Assays, Antitumor/standards , High-Throughput Screening Assays/standards , Humans , Image Processing, Computer-Assisted , Inhibitory Concentration 50 , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Reference Standards , Software
11.
Int J Inflam ; 2012: 505674, 2012.
Article in English | MEDLINE | ID: mdl-22191064

ABSTRACT

The microbiota of the gastrointestinal tract is frequently mentioned as one of the key players in the etiopathogenesis of Crohn's disease (CD). Four hypotheses have been suggested: the single, still unknown bacterial pathogen, an abnormal overall composition of the bowel microbiota ("dysbiosis"), an abnormal immunological reaction to an essentially normally composed microbiota, and increased bacterial translocation. We propose that laser capture microdissection of selected microscopic structures, followed by broad-range 16S rRNA gene sequencing, is an excellent method to assess spatiotemporal alterations in the composition of the bowel microbiota in CD. Using this approach, we demonstrated significant changes of the composition, abundance, and location of the gut microbiome in this disease. Some of these abnormal findings persisted even after macroscopic mucosal healing. Further investigations along these lines may lead to a better understanding of the possible involvement of the bowel bacteria in the development of clinical Crohn's disease.

12.
Neurobiol Learn Mem ; 95(3): 260-9, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21130175

ABSTRACT

Phosphodiesterase 10A (PDE10A) hydrolyzes both cAMP and cGMP, and is a key element in the regulation of medium spiny neuron (MSN) activity in the striatum. In the present report, we investigated the effects of targeted disruption of PDE10A on spatial learning and memory as well as aversive and appetitive conditioning in C57BL/6J mice. Because of its putative role in motivational processes and reward learning, we also determined the expression of the immediate early gene zif268 in striatum and anterior cingulate cortex. Animals showed decreased response rates in scheduled appetitive operant conditioning, as well as impaired aversive conditioning in a passive avoidance task. Morris water maze performance revealed not-motor related spatial learning and memory deficits. Anxiety and social explorative behavior was not affected in PDE10A-deficient mice. Expression of zif268 was increased in striatum and anterior cingulate cortex, which suggests alterations in the neural connections between striatum and anterior cingulate cortex in PDE10A-deficient mice. The changes in behavior and plasticity in these PDE10A-deficient mice were in accordance with the proposed role of striatal MSNs and corticostriatal connections in evaluative salience attribution.


Subject(s)
Association Learning/physiology , Avoidance Learning/physiology , Conditioning, Operant/physiology , Early Growth Response Protein 1/metabolism , Exploratory Behavior/physiology , Phosphoric Diester Hydrolases/physiology , Analysis of Variance , Animals , Appetitive Behavior/physiology , Discrimination, Psychological/physiology , Gyrus Cinguli/metabolism , Gyrus Cinguli/physiology , Male , Maze Learning/physiology , Mice , Mice, Knockout , Neostriatum/metabolism , Neostriatum/physiology , Phosphoric Diester Hydrolases/genetics , Social Behavior , Statistics, Nonparametric
13.
Neuropharmacology ; 58(1): 135-44, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19573542

ABSTRACT

CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression of pro-opiomelanocortin, a key precursor molecule in the hypothalamic pituitary adrenal axis. In AtT20 cells, CRF signalling has been investigated but the mechanism behind CRF-induced ERK activity is not fully understood. This paper elucidates the signalling cascade involved in this phenomenon. Involvement of CRF(1) receptor on ERK phosphorylation was shown by using CRF and urocortin 1. The lack of inhibitory effect of pertussis toxin and BAPTA-AM excluded involvement of G(i)-coupling and calcium mobilization respectively. In contrast, the process is suggested to be driven by cAMP since treatment of AtT20 cells with forskolin triggered strong ERK phosphorylation. Treatment with PKA inhibitors had a minor effect on CRF-induced ERK signalling while phosphorylation of CREB was completely abolished. This ruled out involvement of PKA and suggested a role for exchange protein directly activated by cAMP (EPAC). Moreover, an activator of EPACs 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate mimicked CRF-induced ERK phosphorylation. Gene expression analysis showed high levels of EPAC2 mRNA and protein but low levels of EPAC1. Knockdown of EPAC2 expression by the use of specific siRNAs abolished CRF- and forskolin-induced ERK phosphorylation. The current study demonstrates a clear cAMP-dependent but PKA-independent mechanism underlying CRF-induced ERK activity that proceeds via EPAC2 signalling. Further research will provide more insight in the role of EPAC2 in CRF signalling.


Subject(s)
Carrier Proteins/metabolism , Corticotropin-Releasing Hormone/pharmacology , Cyclic AMP/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Animals , Calcium/metabolism , Carrier Proteins/genetics , Cell Line , Chelating Agents/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanine Nucleotide Exchange Factors/genetics , Humans , Mice , Pertussis Toxin/pharmacology , Phosphorylation/drug effects , RNA, Small Interfering/pharmacology , Receptors, Corticotropin-Releasing Hormone/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Time Factors , Transfection/methods
14.
Clin Gastroenterol Hepatol ; 6(2): 194-205, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18237869

ABSTRACT

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) has been associated with mucosal dysfunction, mild inflammation, and altered colonic bacteria. We used microarray expression profiling of sigmoid colon mucosa to assess whether there are stably expressed sets of genes that suggest there are objective molecular biomarkers associated with IBS. METHODS: Gene expression profiling was performed using Human Genome U133 Plus 2.0 (Affymetrix) GeneChips with RNA from sigmoid colon mucosal biopsy specimens from 36 IBS patients and 25 healthy control subjects. Real-time quantitative polymerase chain reaction was used to confirm the data in 12 genes of interest. Statistical methods for microarray data were applied to search for differentially expressed genes, and to assess the stability of molecular signatures in IBS patients. RESULTS: Mucosal gene expression profiles were consistent across different sites within the sigmoid colon and were stable on repeat biopsy over approximately 3 months. Differentially expressed genes suggest functional alterations of several components of the host mucosal immune response to microbial pathogens. The most strikingly increased expression involved a yet uncharacterized gene, DKFZP564O0823. Identified specific genes suggest the hypothesis that molecular signatures may enable distinction of a subset of IBS patients from healthy controls. By using 75% of the biopsy specimens as a validation set to develop a gene profile, the test set (25%) was predicted correctly with approximately 70% accuracy. CONCLUSIONS: Mucosal gene expression analysis shows there are relatively stable alterations in colonic mucosal immunity in IBS. These molecular alterations provide the basis to test the hypothesis that objective biomarkers may be identified in IBS and enhance understanding of the disease.


Subject(s)
Colon/immunology , Immunity, Mucosal/genetics , Intestinal Mucosa/immunology , Irritable Bowel Syndrome/immunology , Adolescent , Adult , Aged , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Male , Middle Aged , Oligonucleotide Array Sequence Analysis/methods , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods
15.
J Recept Signal Transduct Res ; 27(4): 309-22, 2007.
Article in English | MEDLINE | ID: mdl-17885924

ABSTRACT

The interaction of ghrelin, a 28-residue acylated peptide, with the growth hormone secretagogue receptor 1a (GHS-R1a) has been studied mostly in cells expressing a recombinant GHS-R1a. As awareness is growing on the importance to study G protein-coupled receptors in a natural environment, we studied the effect of ghrelin in the human hepatocellular HepG2 cell line because it has been described in literature to respond to ghrelin. Despite extensive efforts, we were not able to confirm mRNA expression of GHS-R1a by reverse transcription PCR, radioligand binding, or ghrelin-induced GHS-R1a receptor activation; therefore, we conclude that HepG2 cells do not express GHS-R1a. On the other hand, we confirmed a modest effect of ghrelin on the up-regulation of IRS-1 phosphorylation, which might suggest the existence of an alternative ghrelin receptor in HepG2 cells.


Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Calcium/metabolism , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Fluorescent Dyes/pharmacology , Ghrelin , Humans , Insulin Receptor Substrate Proteins , Peptide Hormones/chemistry , Peptide Hormones/metabolism , Phosphoproteins/metabolism , Phosphorylation , Receptors, G-Protein-Coupled/metabolism , Receptors, Ghrelin , Up-Regulation
16.
Gastroenterology ; 132(4): 1375-87, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17408648

ABSTRACT

BACKGROUND & AIMS: Visceral hypersensitivity, a hallmark of irritable bowel syndrome, is generally considered to be mechanosensitive in nature and mediated via spinal afferents. Both stress and inflammation are implicated in visceral hypersensitivity, but the underlying molecular mechanisms of visceral hypersensitivity are unknown. METHODS: Mice were infected with Nippostrongylus brasiliensis (Nb) larvae, exposed to environmental stress and the following separate studies performed 3-4 weeks later. Mesenteric afferent nerve activity was recorded in response to either ramp balloon distention (60 mm Hg), or to an intraluminal perfusion of hydrochloric acid (50 mmol/L), or to octreotide administration (2 micromol/L). Intraperitoneal injection of cholera toxin B-488 identified neurons projecting to the abdominal viscera. Fluorescent neurons in dorsal root and nodose ganglia were isolated using laser-capture microdissection. RNA was hybridized to Affymetrix Mouse whole genome arrays for analysis to evaluate the effects of stress and infection. RESULTS: In mice previously infected with Nb, there was no change in intestinal afferent mechanosensitivity, but there was an increase in chemosensitive responses to intraluminal hydrochloric acid when compared with control animals. Gene expression profiles in vagal but not spinal visceral sensory neurons were significantly altered in stressed Nb-infected mice. Decreased afferent responses to somatostatin receptor 2 stimulation correlated with lower expression of vagal somatostatin receptor 2 in stressed Nb-infected mice, confirming a link between molecular data and functional sequelae. CONCLUSIONS: Alterations in the intestinal brain-gut axis, in chemosensitivity but not mechanosensitivity, and through vagal rather than spinal pathways, are implicated in stress-induced postinflammatory visceral hypersensitivity.


Subject(s)
Brain/physiology , Intestines/innervation , Mesentery/innervation , Nippostrongylus/pathogenicity , Strongylida Infections/metabolism , Visceral Afferents/drug effects , Adjuvants, Immunologic/pharmacology , Animals , Cholera Toxin/pharmacology , Disease Models, Animal , Female , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Ganglia, Spinal/physiopathology , Gene Expression/drug effects , Hydrochloric Acid/pharmacology , Intestinal Mucosa/metabolism , Mesentery/drug effects , Mesentery/metabolism , Mice , Mice, Inbred BALB C , Nodose Ganglion/drug effects , Nodose Ganglion/metabolism , Nodose Ganglion/physiopathology , Octreotide/pharmacology , Polymerase Chain Reaction , RNA/genetics , Receptors, Somatostatin/biosynthesis , Receptors, Somatostatin/genetics , Strongylida Infections/parasitology , Strongylida Infections/pathology , Vagus Nerve/drug effects , Vagus Nerve/metabolism , Vagus Nerve/physiopathology , Visceral Afferents/metabolism
17.
Physiol Genomics ; 24(3): 252-63, 2006 Feb 14.
Article in English | MEDLINE | ID: mdl-16303873

ABSTRACT

Vagal afferent neurons are thought to convey primarily physiological information, whereas spinal afferents transmit noxious signals from the viscera to the central nervous system. To elucidate molecular identities for these different properties, we compared gene expression profiles of neurons located in nodose ganglia (NG) and dorsal root ganglia (DRG) in mice. Intraperitoneal administration of Alexa Fluor-488-conjugated cholera toxin B allowed enrichment for neurons projecting to the viscera. Fluorescent neurons in DRG (from T10 to T13) and NG were isolated using laser-capture microdissection. Gene expression profiles of these afferent neurons, obtained by microarray hybridization, were analyzed using multivariate spectral map analysis, significance analysis of microarrays (SAM) algorithm, and fold-difference filtering. A total of 1,996 genes were differentially expressed in DRG vs. NG, including 41 G protein-coupled receptors and 60 ion channels. Expression profiles obtained on laser-captured neurons were contrasted to those obtained on whole ganglia, demonstrating striking differences and the need for microdissection when studying visceral sensory neurons because of dilution of the signal by somatic sensory neurons. Furthermore, we provide a detailed catalog of all adrenergic and cholinergic, GABA, glutamate, serotonin, and dopamine receptors; voltage-gated potassium, sodium, and calcium channels; and transient receptor potential cation channels present in afferents projecting to the peritoneal cavity. Our genome-wide expression profiling data provide novel insight into molecular signatures that underlie both functional differences and similarities between NG and DRG sensory neurons. Moreover, these findings will offer novel insight into mode of action of pharmacological agents modulating visceral sensation.


Subject(s)
Ganglia, Spinal/metabolism , Gene Expression Profiling/methods , Neurons/metabolism , Nodose Ganglion/metabolism , Peritoneal Cavity/microbiology , Animals , Female , Ganglia, Sensory , Mice , Mice, Inbred BALB C , Peritoneal Cavity/cytology , Signal Transduction
18.
Microbiology (Reading) ; 146 ( Pt 2): 353-365, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10708374

ABSTRACT

Almost all eukaryotic mRNAs are capped at their 5'-terminus. Capping is crucial for stability, processing, nuclear export and efficient translation of mRNA. We studied the phenotypic effects elicited by depleting a Candida albicans strain of mRNA 5'-guanylyltransferase (mRNA capping enzyme; CGT1). Construction of a Cgt1-deficient mutant was achieved by URA-blaster-mediated genetic disruption of one allele of the CGT1 gene, which was localized on chromosome III. The resulting heterozygous mutant exhibited an aberrant colony morphology resembling the 'irregular wrinkle' phenotype typically obtained from a normal C. albicans strain upon mild UV treatment. Its level of CGT1 mRNA was reduced two- to fivefold compared to the parental strain. Proteome analysis revealed a large number of differentially expressed proteins confirming the expected pleiotropic effect of CGT1 disruption. The disrupted strain was significantly more resistant to hygromycin B, an antibiotic which decreases translational fidelity, and showed increased resistance to heat stress. Proteome analysis revealed a 50-fold overexpression of Ef-1alphap and a more than sevenfold overexpression of the cell-wall heat-shock protein Ssa2p. Compared to a reference strain, the cgt1/CGT1 heterozygote was equally virulent for mice and guinea pigs when tested in an intravenous infection model of disseminated candidiasis.


Subject(s)
Anti-Bacterial Agents/pharmacology , Candida albicans/drug effects , Candida albicans/genetics , Gene Deletion , Hygromycin B/pharmacology , Nucleotidyltransferases/genetics , Alleles , Animals , Candida albicans/growth & development , Candida albicans/pathogenicity , Candidiasis/microbiology , Candidiasis/pathology , Chromosome Mapping , Drug Resistance, Microbial/genetics , Electrophoresis, Gel, Two-Dimensional , Genes, Fungal , Guinea Pigs , Heterozygote , Mice , Nucleotidyltransferases/metabolism , Proteome/metabolism , Temperature , Transcription, Genetic , Virulence
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