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Situ Hybridisation (situ + hybridisation)
Terms modified by Situ Hybridisation Selected AbstractsDifferential expression of polycomb repression complex 1 (PRC1) members in the developing mouse brain reveals multiple complexesDEVELOPMENTAL DYNAMICS, Issue 9 2006Tanja Vogel Abstract Polycomb group (PcG) genes are regulators of body segmentation and cell growth, therefore being important players during development. PcG proteins form large complexes (PRC) that fulfil mostly repressive regulative functions on homeotic gene expression. Although expression of PcG genes in the brain has been noticed, the involvement of PcG genes in the processes of brain development is not understood. In this study, we analysed the expression patterns of PRC1 complex members to reveal PcG proteins that might be relevant for mouse brain development. Using in situ hybridisation, we show PRC1 activity in proliferative progenitor cells during neurogenesis, but also in maturated neuronal structures. PRC1 complex compositions vary in a spatial and temporal controlled manner during mouse brain development, providing cellular tools to act in different developmental contexts of cell proliferation, cell fate determination, and differentiation. Developmental Dynamics 235:2574,2585, 2006. © 2006 Wiley-Liss, Inc. [source] Definition and spatial annotation of the dynamic secretome during early kidney developmentDEVELOPMENTAL DYNAMICS, Issue 6 2006Gemma Martinez Abstract The term "secretome" has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350,1359). The term "secreted protein" encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants. Developmental Dynamics 235:1709,1719, 2006. © 2006 Wiley-Liss, Inc. [source] Cranial expression of class 3 secreted semaphorins and their neuropilin receptorsDEVELOPMENTAL DYNAMICS, Issue 4 2003John K. Chilton Abstract The semaphorin family of chemorepellents and their receptors the neuropilins are implicated in a variety of cellular processes, including axon guidance and cell migration. Semaphorins may bind more than one neuropilin or a heterodimer of both, thus a detailed knowledge of their expression patterns may reveal possible cases of redundancy or mutual antagonism. To assess their involvement in cranial development, we cloned fragments of the chick orthologues of Sema3B and Sema3F. We then carried out mRNA in situ hybridisation of all six class 3 semaphorins and both neuropilins in the embryonic chick head. We present evidence for spatiotemporal regulation of these molecules in the brainstem and developing head, including the eye, ear, and branchial arches. These expression patterns provide a basis for functional analysis of semaphorins and neuropilins in the development of axon projections and the morphogenesis of cranial structures. Developmental Dynamics 228:726,733, 2003. © 2003 Wiley-Liss, Inc. [source] Sorting nexin-14, a gene expressed in motoneurons trapped by an in vitro preselection methodDEVELOPMENTAL DYNAMICS, Issue 4 2001Patrick Carroll Abstract A gene-trap strategy was set up in embryonic stem (ES) cells with the aim of trapping genes expressed in restricted neuronal lineages. The vector used trap genes irrespective of their activity in undifferentiated totipotent ES cells. Clones were subjected individually to differentiation in a system in which ES cells differentiated into neurons. Two ES clones in which the trapped gene was expressed in ES-derived neurons were studied in detail. The corresponding cDNAs were cloned, sequenced, and analysed by in situ hybridisation on wild-type embryo sections. Both genes are expressed in the nervous system. One gene, YR-23, encodes a large intracellular protein of unknown function. The second clone, YR-14, represents a sorting nexin (SNX14) gene whose expression in vivo coincides with that of LIM-homeodomain Islet-1 in several tissues. Sorting nexins are proteins associated with the endoplasmic reticulum (ER) and may play a role in receptor trafficking. Gene trapping followed by screening based on in vitro preselection of differentiated ES recombinant clones, therefore, has the potential to identify integration events in subsets of genes before generation of mouse mutants. © 2001 Wiley-Liss, Inc. [source] Detection of EHV-1 and EHV-4 in placental sections of naturally occurring EHV-1- and EHV-4-related abortions in the UK: use of the placenta in diagnosisEQUINE VETERINARY JOURNAL, Issue 5 2003S. GERST Summary Reasons for performing study: EHV-1 and EHV-4 abortion diagnosis is based upon detailed examination of the aborted fetus. However, in some cases, only the placenta is available for examination. Furthermore, the contribution of lesions in the placenta to pathogenesis and diagnosis of EHV-1 and EHV-4 abortion has been neglected. Objectives: To assess the utility of placental examination in equine herpesvirus-1 (EHV-1) and EHV-4 abortion diagnosis. Methods: Sections of allantochorion from 49 herpesvirus abortions were analysed by PCR, in situ hybridisation and immunostaining. Results: Virus-specific nested PCR confirmed the presence of viral DNA in 46 cases; 41 cases were EHV-1-positive and 5 EHV-4-positive. Microscopic changes were nonspecific. Examination of the PCR-positive sections of allantochorion revealed EHV-1 DNA by in situ hybridisation (ISH) in 21 cases and EHV-4 in 4 cases. In 2 samples, DNA of both viruses was present on PCR and ISH. Viral antigen was found by immunohistology in 15 cases. Regarding the localisation of virus in the placentae, both viral DNA and antigen of EHV-1 and EHV-4 were found in endothelial cells of chorionic villi and, occasionally, in trophoblast epithelium. In the stromal endothelium, only EHV-1 was found. Conclusions: The data indicate that examination of placentae is a useful diagnostic aid in EHV-1 and EHV-4 abortion diagnosis. Potential relevance: Virological examination of the placenta should be come standard practice in equine abortion investigations, particularly in those cases where the fetus is not available for examination. [source] A narrow deletion of 7q is common to HCL, and SMZL, but not CLLEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2004Claus Lindbjerg Andersen Abstract: To further characterise the genetic background of the two closely related B-lymphocytic malignancies hairy cell leukaemia (HCL), and splenic marginal zone lymphoma (SMZL) we have identified characteristic copy number imbalances by comparative genomic hybridisation (CGH). Based on these findings, areas of special interest were fine mapped, and relevant probes constructed for use in interphase-fluorescence in situ hybridisation (FISH) investigations. Thus, using the CGH data from 52 HCL and 61 SMZL patients, we identified the characteristic profiles of copy number imbalances for both diseases. These were a gain of 5q13-31 (19%) and loss of 7q22-q35 (6%) for HCL, and gain of 3q25 (28%), loss of 7q31 (16%), and gain of 12q15 (16%) for SMZL. A partial loss of 7q unsual for low-malignant B-cell diseases was found to be common to the two diseases. This loss was therefore fine mapped with BAC/PAC clones. Fine mapping revealed that in SMZL the minimal lost region covers 11.4 Mb spanning from 7q31.33 to 7q33 located between sequence tagged site (STS)-markers SHGC-3275 and D7S725. This area was distinct from the commonly deleted 7q region of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML). A FISH probe specific for the 7q region was constructed. Using this probe in an interphase-FISH investigation we showed the minimal lost 7q-region of HCL and SMZL to be one and the same. In one HCL case, this investigation furthermore showed the extent of the deleted region to be below the detection limit of CGH, whereas interphase-FISH screening of 36 chronic lymphocytic leukaemia (CLL) cases showed no deletion of the 7q area. In conclusion, we have identified characteristic profiles of copy number imbalances in HCL and SMZL and fine mapped the minimal extent of a commonly lost 7q area of special interest. We hypothesise that this region may contain (a) gene(s) important for the pathology of HCL and SMZL. [source] Characterisation of microbial community composition of a Siberian tundra soil by fluorescence in situ hybridisationFEMS MICROBIOLOGY ECOLOGY, Issue 1 2004Svenja Kobabe Abstract The bacterial community composition of the active layer (0,45 cm) of a permafrost-affected tundra soil was analysed by fluorescence in situ hybridisation (FISH). Arctic tundra soils contain large amounts of organic carbon, accumulated in thick soil layers and are known as a major sink of atmospheric CO2. These soils are totally frozen throughout the year and only a thin active layer is unfrozen and shows biological activity during the short summer. To improve the understanding of how the carbon fluxes in the active layer are controlled, detailed analysis of composition, functionality and interaction of soil microorganisms was done. The FISH analyses of the active layer showed large variations in absolute cell numbers and in the composition of the active microbial community between the different horizons, which is caused by the different environmental conditions (e.g., soil temperature, amount of organic matter, aeration) in this vertically structured ecosystem. Universal protein stain 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) showed an exponential decrease of total cell counts from the top to the bottom of the active layer (2.3 × 109,1.2 × 108 cells per gram dry soil). Using FISH, up to 59% of the DTAF-detected cells could be detected in the surface horizon, and up to 84% of these FISH-detected cells could be affiliated to a known phylogenetic group. The amount of FISH-detectable cells decreased with increasing depth and so did the diversity of ascertained phylogenetic groups. [source] Loss of Hypothalamic Response to Leptin During Pregnancy Associated with Development of Melanocortin ResistanceJOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2009S. R. Ladyman Hypothalamic leptin resistance during pregnancy is an important adaptation that facilitates the state of positive energy balance required for fat deposition in preparation for lactation. Within the arcuate nucleus, pro-opiomelanocortin (POMC) neurones and neuropeptide Y (NPY)/agouti-related gene protein (AgRP) neurones are first-order leptin responsive neurones involved in the regulation of energy balance. The present study aimed to investigate whether the regulation of these neuropeptides is disrupted during pregnancy in association with the development of leptin resistance. As measured by quantitative in situ hybridisation, POMC and AgRP mRNA levels were not significantly different during pregnancy, whereas NPY mRNA levels increased such that, by day 21 of pregnancy, levels were significantly higher than in nonpregnant, animals. These data suggest that these neurones were not responding normally to the elevated leptin found during pregnancy. To further characterise the melanocortin system during pregnancy, double-label immunohistochemistry was used to quantify leptin-induced phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in POMC neurones, using ,-melanocyte-stimulating hormone (MSH) as a marker. The percentage of ,-MSH neurones containing leptin-induced pSTAT3 did not significantly differ from nonpregnant animals, indicating that there was no change in the number of POMC neurones that respond to leptin during pregnancy. Treatment with ,-MSH significantly reduced food intake in nonpregnant rats, but not in pregnant rats, indicating resistance to the satiety actions of ,-MSH during pregnancy. The data suggest that multiple mechanisms contribute to leptin resistance during pregnancy. As well as a loss of responses in first-order leptin-responsive neurones in the arcuate nucleus, there is also a downstream disruption in the melanocortin system. [source] Serotonergic and Catecholaminergic Interactions with Co-Localised Dopamine-Melatonin Neurones in the Hypothalamus of the Female TurkeyJOURNAL OF NEUROENDOCRINOLOGY, Issue 1 2009S. W. Kang Serotonin and catecholamines (dopamine, norepinephrine, epinephrine) have important roles as neurotransmitters in avian reproduction, but their anatomical relationship to the neuroendocrine circuitry that regulates reproduction is poorly understood. Our previous studies have shown that co-localised dopamine-melatonin (DA-MEL) neurones in the avian premammillary nucleus (PMM) are active during periods of photoresponsiveness and, therefore, are potentially photosensitive neurones. Because serotonergic and catecholaminergic neurotransmitters are important regulators of reproductive function in the female turkey, we hypothesised that the serotonergic/catecholaminergic neurones within the brainstem might interact with PMM DA-MEL neurones and constitute an important circuit for reproductive function. To examine this possible interaction, the retrograde fluorescent tract tracer, 1,1,dioctadecyl-3,3,3,3,-tetramethyleindocarbocyanine perchlorate (DiI) was injected into the PMM, and combined with serotonin, tyrosine hydroxylase (TH), dopamine ,-hydroxylase (DBH) and phenyl N -methyltransferse (PNMT) immunocytochemistry to reveal neuroanatomical connections. Changes in the activities of serotonergic, dopaminergic, adrenergic and noradrenergic neuronal systems projecting to the PMM were measured at different reproductive states with in situ hybridisation (ISH) techniques, using tryptophan hydroxylase 2 (TPH2) and TH mRNA expression, respectively. Cells labelled with DiI were found in anatomically discrete areas in or near the hypothalamus and the brainstem. Double immunocytochemistry confirmed that there were serotonin, DBH and PNMT fibres in close apposition to DA-MEL neurones. TPH2 mRNA expression in serotonin neurones was found in several nuclei, and its most abundant mRNA expression was seen in the nucleus Locus ceruleus of laying and incubating hens. TH mRNA expression levels in the six catecholaminegic areas labelled with DiI was measured across the different reproductive states. In the nucleus tractus solitarius (adrenergic), the highest level of TH mRNA expression was found in photorefractory hens and the lowest level in incubating hens. These observed patterns of serotonin/catecholamine neuronal distribution and their variable interactions with PMM DA-MEL neurones during different reproductive states may offer a significant neuroanatomical basis for understanding the control of avian reproductive seasonality. [source] Rhythm-Dependent Light Induction of the c-fos Gene in the Turkey HypothalamusJOURNAL OF NEUROENDOCRINOLOGY, Issue 6 2007A. Thayananuphat Day length (photoperiod) is a powerful synchroniser of seasonal changes in the reproductive neuroendocrine activity in temperate-zone birds. When exposed to light during the photoinducible phase, reproductive neuroendocrine responses occur. However, the neuroendocrine systems involved in avian reproduction are poorly understood. We investigated the effect of light exposure at different circadian times upon the hypothalamus and components of the circadian system, using c-fos mRNA expression, measured by in situ hybridisation, as an indicator of light-induced neuronal activity. Levels of c-fos mRNA in these areas were compared after turkey hens (on a daily 6-h light period) had been exposed to a 30-min period of light occurring at 8, 14, or 20 h after the onset of first light of the day (subjective dawn). Non-photostimulated control birds were harvested at the same times. In birds, photostimulated within the photoinducibile phase (14 h), in contrast to before or after, c-fos mRNA was significantly increased in the nucleus commissurae pallii (nCPa), nucleus premamillaris (PMM), eminentia mediana (ME), and organum vasculosum lamina terminalis (OVLT). Photostimulation increased c-fos mRNA expression in the pineal gland, nucleus suprachiasmaticus, pars visualis (vSCN) and nucleus inferioris hypothalami compared to that of their corresponding nonphotostimulated controls. However, the magnitudes of the responses in these areas were similar irrespective of where in the dark period the pulses occurred. No c-fos mRNA was induced in the nucleus infundibulari, in response to the 30-min light period at any of the circadian times tested. The lack of c-fos up-regulation in the pineal gland and vSCN following photostimulation during the photoinducible phase lends credence to the hypothesis that these areas are not involved in the photic initiation of avian reproduction. On the other hand, c-fos mRNA increases in the nCPa, ME, and OVLT support other studies showing that these areas are involved in the onset of reproductive behaviour initiated by long day lengths. The present study provides novel data showing that the PMM in the caudal hypothalamus is involved in the neuronally mediated, light-induced initiation of reproductive activity in the turkey hen. [source] Altered Expression of SOCS3 in the Hypothalamic Arcuate Nucleus during Seasonal Body Mass Changes in the Field Vole, Microtus agrestisJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2007E. Król We have previously shown that cold-acclimated (8 °C) male field voles (Microtus agrestis) transferred from short day (SD, 8 h light) to long day (LD, 16 h light) photoperiod exhibit an increase in body mass lasting 4 weeks, after which they stabilise at a new plateau approximately 7.5 g (24.8%) higher than animals maintained in SD. By infusing voles with exogenous leptin, we have also demonstrated that SD voles respond to the hormone by reducing body mass and food intake, whereas LD animals increasing body mass are resistant to leptin treatment. In the present study, we investigated whether seasonal changes in body mass could be linked to modulation of the leptin signal by suppressor of cytokine signalling-3 (SOCS3). We used in situ hybridisation to examine hypothalamic arcuate nucleus (ARC) expression of SOCS3, neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) genes in 90 voles exposed to either SD or LD for up to 11 weeks. LD voles increasing body mass had significantly higher levels of SOCS3 mRNA than SD or LD voles with a stable body mass. There were no associated changes in expression of NPY, AgRP, POMC and CART genes. These results suggest that voles that regulate body mass at either the lower (SD) or upper (LD) plateau remain sensitive to leptin action, whereas SOCS3-mediated leptin resistance is a short-term mechanism that enables animals to move between the stable body mass plateaus. Our data provide evidence that expression of SOCS3 in the ARC is involved in the modulation of the strength of the leptin signal to facilitate seasonal cycles in body mass and adiposity. [source] Stroke Injury in Rats Causes an Increase in Activin A Gene Expression Which is Unaffected by Oestradiol TreatmentJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2006M. Böttner Abstract Activins are members of the transforming growth factor-, superfamily that exert neurotrophic and neuroprotective effects on various neuronal populations. To determine the possible function of activin in stroke injury, we assessed which components of the activin signalling pathway were modulated in response to middle cerebral artery occlusion (MCAO). Furthermore, because oestradiol replacement protects against MCAO-induced cell death, we explored whether oestradiol replacement influences activin gene expression. Female Sprague-Dawley rats underwent permanent MCAO and the expression of activins and their corresponding receptors was determined by semiquantitative reverse transcriptase-polymerase chain reaction at 24 h after onset of ischaemia. We observed up-regulation of activin ,A and activin type I receptor A mRNA in response to injury. Dual-label immunocytochemistry followed by confocal z-stack analysis showed that the activin A expressing cells comprised neurones. Next, we monitored the time course of activin ,A mRNA expression in oestradiol- or vehicle-treated rats at 4, 8, 16 and 24 h after MCAO via in situ hybridisation. Starting at 4 h after injury, activin ,A mRNA was up-regulated in cortical and striatal areas in the ipsilateral hemisphere. Activin ,A mRNA levels in the cortex increased dramatically with time and were highest at 24 h after the insult, and oestradiol replacement did not influence this increase. [source] Noradrenergic Regulation of Hypothalamic Cells that Produce Growth Hormone-Releasing Hormone and Somatostatin and the Effect of Altered Adiposity in SheepJOURNAL OF NEUROENDOCRINOLOGY, Issue 6 2005J. Iqbal Abstract The growth hormone (GH) axis is sensitive to alteration in body weight and there is evidence that central noradrenergic systems regulate neurones that produce growth hormone-releasing hormone (GHRH) and somatostatin (SRIF). This study reports semiquantitative estimates of the noradrenergic input to neuroendocrine GHRH and SRIF neurones in the sheep of different body weights. We also studied the effects of altered body weight on expression of dopamine ,-hydroxylase (DBH), the enzyme that produces noradrenalin from dopamine. Ovariectomised ewes were made Lean (39.6 ± 2.6 kg; Mean ± SEM) by dietary restriction, whereas Normally Fed animals (61.2 ± 0.8 kg) were maintained on a regular diet. Brains were perfused for immunohistochemistry and in situ hybridisation. The Mean ± SEM number of GHRH-immunoreactive (-IR) cells was lower in Normally Fed (65 ± 7) than in Lean (115 ± 14) animals, whereas the number of SRIF-IR cells was similar in the two groups (Normally Fed, 196 ± 17; Lean 230 ± 21). Confocal microscopic analysis revealed that the percentage of GHRH-IR cells (Normally Fed 36 ± 1.5% versus Lean 32 ± 4.6%) and percentage of SRIF-IR cells (Normally Fed 30 ± 40.4% versus Lean 32 ± 2.3%) contacted by noradrenergic fibres did not change with body weight. FluoroGold retrograde tracer injections confirmed that noradrenergic projections to the arcuate nucleus are from ventrolateral medulla and noradrenergic projections to periventricular nucleus arise from the ventrolateral medulla, nucleus of solitary tract, locus coeruleus (LC) and the parabrachial nucleus (PBN). DBH expressing cells were identified using immunohistochemistry and in situ hybridisation and the level of expression (silver grains/cell) quantified by image analysis. The number of DBH cells was similar in Normally Fed and Lean animals, but the level of expression/cell was lower (P < 0.02) in the PBN and LC of Lean animals. These results provide an anatomical basis for the noradrenergic regulation of GHRH and SRIF cells and GH secretion. Altered activity or noradrenergic neurones in the PBN and LC that occur with reduced body weight may be relevant to the control of GH axis. [source] Introduction of a High-Energy Diet Acutely Up-Regulates Hypothalamic Cocaine and Amphetamine-Regulated Transcript, Mc4R and Brown Adipose Tissue Uncoupling Protein-1 Gene Expression in Male Sprague-Dawley RatsJOURNAL OF NEUROENDOCRINOLOGY, Issue 1 2005Z. A. Archer Abstract Obesity is an escalating problem in Western societies. Susceptibility to weight gain within an obesogenic environment is variable. It remains unclear how the range of weight gain responses are generated. It is possible that an individual's immediate and/or sustained appetite for apparently palatable foods, or metabolic adaptations to a new diet could be important. The present study therefore examined the short- to medium-term effects of a high-energy (HE) diet on bodyweight, food intake, and energy balance-related signalling systems. Sprague-Dawley rats were fed either chow or an HE diet for 12 h, 24 h, 48 h or 14 days. Blood hormones and metabolites were assayed, and expression of uncoupling protein-1 (UCP-1) and hypothalamic energy-balance related genes were determined by Northern blotting or in situ hybridisation, respectively. Short-term exposure (12 h, 24 h, 48 h) to the HE diet had no effect on grams of food consumed, but caloric intake was increased. Exposure to HE diet for 14 days (medium term) established a bodyweight differential of 7.7 g, and animals exhibited a transient increase in caloric intake of 5 days duration. Terminal levels of leptin, insulin, glucose and non-esterified fatty acids (NEFAs) were all increased in HE-fed animals. UCP-1 mRNA was elevated in interscapular brown adipose tissue from HE-fed rats only at 12 h. Cocaine and amphetamine-regulated transcript (CART) and Mc4R gene expression in the hypothalamus were increased after 12 h and 24 h on an HE diet, respectively. The rats appear to passively over-consume calories as a result of consuming a similar weight of a more energy dense food. This evokes physiological responses, which adjust caloric intake over several days. Circulating NEFA and insulin concentrations, UCP-1, Mc4R and CART gene expression are increased as an immediate consequence of consuming HE diet, and may be involved in countering hypercaloric intake. Circulating leptin is increased in the HE-fed animals after 48 h, reflecting their increasing adiposity. [source] Absence of leukocyte microchimerism in oral lichen planus (OLP): an in situ hybridisation studyJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 7 2001T. Lombardi Abstract: Oral lichen planus (OLP) is a relatively common chronic inflammatory disease. The majority of patients are between 30 and 50 years of age with a higher incidence in females. The aetiology is unknown and various hypotheses on the pathogenic mechanisms, including autoimmunity, have been proposed over the years. In the present study, we investigated whether leukocyte microchimerism, a biological situation implicated in the aetiology of some autoimmune diseases, might play a role in the pathogenesis of OLP. We used in situ hybridisation to identify Y chromosome DNA in a series of formalin-fixed paraffin-embedded oral mucosa biopsies of women with established clinical and histological disease who had given birth to a male child. The positive control, two mucosal specimens from a man with OLP, showed over 90% of keratinocytes and cells within the inflammatory infiltrate, a positive nuclear signal. The negative control, biopsies from three women having carried only female foetuses and one nulliparous woman, all with OLP, did not show any nuclear signal. In the fifteen selected cases of OLP biopsies from women who had only male offspring, nucleated cells containing the Y chromosome were not detected within the chronic inflammatory infiltrate. These results suggest that unlike some other immunologically mediated diseases, leukocyte microchimerism does not seems to be involved in the pathogenesis of OLP. [source] Ethanol and red wine polyphenols induce the short-term downregulation of PAI-1 gene expression in vivo in rat aortic endotheliumJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 9 2007Hernan E Grenett Abstract Moderate alcohol or red wine consumption reduces the risk of cardiovascular mortality. This cardiovascular protection is likely due to the additive, combined and/or synergistic effects of alcohol itself or other components of wine, in particular polyphenols. Experiments were carried out to determine whether ethanol/polyphenols also decrease plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in vivo, using the rat as an animal model. Male Sprague,Dawley rats were gavaged with ethanol, the individual polyphenols catechin and quercetin or saline vehicle. The in vivo effect of ethanol or individual polyphenols on PAI-1 mRNA was then assessed by in situ hybridisation and quantitative reverse transcriptase (RT) polymerase chain reaction (RT-PCR). PAI-1 mRNA expression was significantly reduced in the endothelial and smooth muscle cells of the thoracic aorta of all experimental rats. RT-PCR analysis of PAI-1 mRNA levels in vascular tissue showed a ,55% reduction in PAI-1 mRNA consistent with the decrease in aortic endothelium PAI-1 mRNA observed with in situ hybridisation. This decrease may enhance endothelial cell (EC)-mediated fibrinolytic activity in vivo. The cardioprotection afforded by moderate red wine consumption can therefore be attributed in part to the combined effects of ethanol and individual polyphenols on EC fibrinolysis. Copyright © 2007 Society of Chemical Industry [source] The lobular expression of the rat asialoglycoprotein receptor is regulated at the posttranscriptional levelLIVER INTERNATIONAL, Issue 1 2005Mara Massimi Abstract: The purpose of this study was to define the distribution of the asialoglycoprotein receptor (ASGP-R) main peptide, rat hepatic lectin (RHL)-1, within the rat liver lobule and to investigate its possible modulation in physiological states characterised by marked changes of receptorial expression. In particular, we chose livers from rats partially hepatectomised or at the end of pregnancy, as models, respectively, of decreased or increased expression of the ASGP-R, and used the in situ hybridisation and immunocytochemistry techniques to analyse in parallel the lobular distributions of RHL-1 mRNA and protein. In normal rat liver, although the RHL-1 mRNA was homogeneously distributed, the RHL-1 peptide was predominantly localised on the surface of pericentral hepatocytes with a gradient of expression towards the periportal zone. This gradient of expression of RHL-1 peptide was reduced in regenerating livers, in which the positive stain was restricted to a few layers of cells around the central vein. In contrast, livers at the end of pregnancy showed an overall increase of the peptide with a concomitant flattening of the gradient across the liver plate. In all the conditions, we never observed important changes in the pattern of expression of the specific mRNA. These findings indicate that the distribution of ASGP-R is heterogeneous across the liver lobule, with a pattern of expression prevalently modulated at the posttranscriptional level. [source] Non-radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heartMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2002Arnoud C. Fijnvandraat Abstract Non-radioactive in situ hybridisation is an excellent method to visualise mRNA molecules within their topographical context. Recently we have reported a new non-radioactive in situ hybridisation procedure on tissue sections that is essentially based on the whole mount in situ hybridisation procedure. This method is superior in spatial resolution and sensitivity compared to the radioactive in situ hybridisation procedure. Generally, low levels of gene expression, such as found with the developmental onset of gene expression and in differentiating embryonic stem cells, are difficult to detect by in situ hybridisation. Here an application of the protocol is presented which is based on tyramide signal amplification, which enables the detection of very low abundant mRNAs. The significance of this method is two-fold: (1) the molecular phenotype of embryonic stem cell-derived cardiomyocytes can be examined at the cellular level with high sensitivity, and (2) the number of cells that express the gene of interest can be assessed. Microsc. Res. Tech. 58:387,394, 2002. © 2002 Wiley-Liss, Inc. [source] Prenatal diagnosis of partial tetrasomy 14: a case studyPRENATAL DIAGNOSIS, Issue 2 2002Alice M. George Abstract Prenatal specimens were received from a fetus with abnormalities noted on ultrasound. A supernumerary marker chromosome (SMC) was detected: 47,XY,+mar. Fluorescence in situ hybridisation (FISH) further classified this to be partial tetrasomy for chromosome 14. We compare this finding with other cases of SMC (14) and further classify phenotype with karyotype. Copyright © 2002 John Wiley & Sons, Ltd. [source] Identification of a small supernumerary marker chromosome, r(2)(p10q11.2), and the problem of determining prognosisPRENATAL DIAGNOSIS, Issue 10 2001N. Villa Abstract The identification of small supernumerary marker chromosomes (SMCs) and the elucidation of their clinical significance remain two of the problems in classical human cytogenetics. We observed a small supernumerary ring in amniotic fluid cell cultures and identified its origin as r(2)(p10q11.2) and its extent by means of fluorescent in situ hybridisation (FISH). Uniparental disomy (UPD) was excluded by microsatellite analysis using polymorphic markers localised in the same region. On the basis of normal ultrasonographic checks, the patient decided to continue the pregnancy. A normal female was delivered at term and subsequent neonatal follow-ups confirmed the normal phenotype and development. In the present case, genetic counselling was not helpful because of the absence of reference cases. Detailed characterisation made it possible to correlate the normal baby phenotype with the trisomic 2p10-2q11.2 genomic region. Further molecular cytogenetic investigations of SMCs classified by DNA content and pregnancy outcome data should improve genetic counselling and risk evaluation. Copyright © 2001 John Wiley & Sons, Ltd. [source] A study of meiotic segregation of chromosomes in spermatozoa of translocation carriers using fluorescent in situ hybridisationANDROLOGIA, Issue 1 2010A. Perrin Summary In the infertile male population, there is a 2,20-time higher probability of having a structural chromosomal abnormality than in general population. Generally, these men have a normal phenotype but they can have sperm abnormalities. As they can produce a variable proportion of unbalanced gametes, it is important to evaluate the percentage of unbalanced chromosomal spermatozoa to assess the risk of injecting a chromosomally unbalanced gamete during ICSI procedure. We report here the meiotic segregation analysis of chromosomes in spermatozoa of 12 men with a balanced reciprocal translocation and 4 men with a Robertsonian translocation using a fluorescent in situ hybridisation analysis. The frequencies of normal or balanced spermatozoa ranged from 34.4% to 49.1% in balanced reciprocal translocation carriers. For Robertsonian translocation, the frequencies of normal or balanced spermatozoa ranged from 78.4% to 91.2%. These analyses allow us to define the orientation of genetic counselling according to the results of meiotic segregation obtained. As a last resort, it could then be discussed of the possibility of having recourse to donor spermatozoa or adoption. [source] TEM and FISH studies in sperm from men of couples with recurrent pregnancy lossANDROLOGIA, Issue 6 2009G. Collodel Summary The role of the male partner in recurrent pregnancy loss (RPL) is not clear. In this study, semen characteristics of 22 men whose partners had experienced RPL were examined by light microscopy. Sperm morphology was analysed by transmission electron microscopy (TEM) and the data were mathematically elaborated to obtain numerical indices expressing the status of an ejaculate: the fertility index and the percentage of apoptosis, necrosis and immaturity. Sperm apoptosis and necrosis were also evaluated by annexin V/propidium iodide assay. To explore the status of meiotic segregation, fluorescence in situ hybridisation (FISH) with probes for chromosomes 18, X and Y, was applied directly on sperm nuclei. Sperm characteristics from a group of men of proven fertility were used as controls. Among the considered sperm characteristics, apoptosis (P < 0.01), 1818YY diploidy (P < 0.05) and 18YY disomy (P < 0.01) scores were significantly higher in men with RPL compared with controls. Our study showed that some patients with normal semen parameters can present a slight increase in aneuploidy compared with controls, indicating a possible involvement of sperm in some cases of RPL. Chromosomal FISH analysis and chromatin tests of sperm could be included in RPL work-ups when no other cause has been detected. [source] Comparison of semen profile and frequency of chromosome aneuploidies in sperm nuclei of patients with varicocele before and after varicocelectomyANDROLOGIA, Issue 3 2009H. Acar Summary Semen profile and meiotic segregation products are important for assessing aneuploidy risk and risk of resulting infertility. To determine the effect of varicocelectomy on semen profile and aneuploidy frequency, we investigated semen profile and aneuploidy frequency of selected chromosomes in patients with varicocele before and after varicocelectomy. Chromosomal aneuploidy for selected chromosomes was evaluated using chromosome-specific DNA fluorescence in situ hybridisation (FISH) probes. There was a significant difference in the level of normal sperm morphology before and after varicocelectomy (P > 0.007). There were no significant differences in aneuploidy frequency of chromosomes 1, 16, 17 and 18 in sperm nuclei obtained from patients before varicocelectomy compared with 6,7 months after varicocelectomy (P > 0.05), although FISH analysis with chromosomes 17 and 18 combination showed a higher aneuploidy frequency before varicocelectomy than after varicocelectomy (7.81 ± 9.67 versus 4.03 ± 1.46 respectively). In conclusion, varicocele seems to affect the semen profile but minimally affects aneuploidy frequency. Varicocelectomy demonstrates a repairing effect on the semen profile and contributes to a slight decrease in aneuploidy frequency in some but not all chromosomes. [source] Identification and expression analysis of a MYB family transcription factor in the parasitic plant Orobanche ramosaANNALS OF APPLIED BIOLOGY, Issue 2 2007C.I. González-Verdejo Abstract MYB proteins are transcription factors (TFs) involved in the regulation of developmental processes in eukaryotes. A number of MYB genes have been identified from plants, but they have not been studied in parasitic plants. In this work, a member of the R2R3 MYB family of TFs was isolated from a complementary DNA library representing different developmental stages of the parasitic plant Orobanche ramosa. The pattern of expression of the gene was studied by in situ hybridisation. Alignment of the deduced Or-MYB1 protein with members of the MYB family showed the highest overall identity with MYB.Ph3 from petunia (Petunia hybrida), NtMYBAS1/S2 from tobacco (Nicotiana tabacum) and AtMYB101 from Arabidopsis thaliana. Amino acid sequence comparisons of DNA-binding domains showed that Or-MYB1 protein forms a closely related group with these proteins. Transcripts of Or-MYB1 were detected during all the developmental stages analysed, and in situ hybridisation showed that the expression was restricted to the parenchymatic cells proximal to the vascular vessels. These findings are consistent with a role of Or-MYB1 during early stages of development of O. ramosa, probably through the phenylpropanoid pathway. [source] Characterisation of distant Alstrogmeria hybrids: application of highly repetitive DNA sequences from A. ligtu ssp. ligtuANNALS OF APPLIED BIOLOGY, Issue 3 2003SHUJUN ZHOU Summary Clones from a Sau 3A family of eight highly repetitive sequences previously isolated from a genomic DNA library of Alstroemeria ligtu ssp. ligtu were sequenced and found to be highly conserved. A trinucleotide microsatellite repeat [GCA]3,4 was present. A second, unrelated, Sau 3A repeat was also characterised. Southern analysis proved that the isolated repeats were specific for the A. ligtu subspecies and could not be detected in other Chilean or Brazilean Alstroemeria species. As shown by in situ hybridisation, the Sau 3A family and the unrelated Sau 3A repeat co-localised at distinct sites along most chromosomes of Alstroemeria ligtu ssp. ligtu and Alstroemeria ligtu ssp. simsii. The present set of species-specific repetitive sequences enables the identification of A. ligtu chromosomes, and thus the tracking of chromosome transmission to interspecific hybrids and their progeny. [source] In situ hybridisation for identification and differentiation of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae and Mycoplasma hyorhinis in formalin-fixed porcine tissue sections,APMIS, Issue 10 2001M. BOYE Oligonucleotide probes targeting 16S ribosomal RNA were designed for species-specific identification of the porcine mycoplasmas Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma hyosynoviae using a fluorescent in situ hybridisation assay. The specificity of the probes was evaluated using pure cultures as well as porcine tissue sections with artificial presence of mycoplasma, and the probes were found specific for the target organisms. The assay was applied on sections of 28 tissue samples from pigs infected with one or more of the three Mycoplasma species as determined by cultivation. M. hyopneumoniae and M. hyorhinis were identified in accordance with cultivation in lung sections from nine pigs affected by catarrhal to purulent bronchopneumonia. Likewise, in eight cases of fibrinous pericarditis, M. hyopneumoniae, M. hyorhinis and M. hyosynoviae were the infectious agents according to cultivation and were correctly identified by in situ hybridisation. Out of 11 joints cultivation positive for M. hyosynoviae, the probe was only able to identify M. hyosynoviae in eight cases probably due to a low number of microorganisms in the tissue sections. The in situ hybridisation assay is well suited for use in diagnostic and experimental work as well as a tool for pathogenesis studies. [source] Original Article: Rate of X chromosome aneuploidy in young fertile women: Comparison of cultured and uncultured cell preparations using fluorescence in situ hybridisationAUSTRALIAN AND NEW ZEALAND JOURNAL OF OBSTETRICS AND GYNAECOLOGY, Issue 4 2010Kirralee PATTON Background:, X chromosome aneuploidy <10% in female patients is a routinely used reporting limit in diagnostic cytogenetics. X aneuploidy (<10%) is commonly detected in women investigated for infertility or recurrent miscarriages. It is unclear if this aneuploidy is causally relevant or related to the culture process. Information about the background rate of X aneuploidy in young fertile women would be helpful in resolving this issue. Aim:, This study aimed to investigate the rate of X aneuploidy in young fertile women in cultured and uncultured samples to determine if the commonly used <10% limit is relevant. Method:, Volunteers (aged 22,40 years) with proven fertility (n = 78) participated. The number of X chromosome signals in 500 cultured and 500 uncultured preparations were enumerated using FISH. Results:, Significantly, all participants had <5% X aneuploidy in both preparations, X chromosome loss occurred (2.4%) more frequently than gain (0.7%). Cultured preparations had a mean of 2.1% cells with X chromosome aneuploidy (95% CI 1.9,2.3%) compared with a mean rate of 0.9% aneuploidy in uncultured preparations (95% CI 0.7,1.1%). The relative risk for cultured preparations having X aneuploidy compared with uncultured cells was 2.33 (P < 0.001) (95% CI 2.1,2.6). Conclusion:, Young fertile women had <5% X aneuploidy. The rate of X aneuploidy was higher in cultured (2.1%) compared with uncultured (0.9%) preparations (P < 0.001). This data may provide useful background information when considering low level X aneuploidy in other groups of women with clinical indications for karyotype. [source] Oxytocin mRNA content in the endometrium of non-pregnant womenBJOG : AN INTERNATIONAL JOURNAL OF OBSTETRICS & GYNAECOLOGY, Issue 3 2004Margareta Steinwall Objective To study oxytocin mRNA in the human endometrium at different phases of the menstrual cycle. Design An exploratory study in non-pregnant women. Setting The Department of Obstetrics and Gynecology, Lund University Hospital, Sweden. Participants Thirty-three women of fertile age undergoing hysterectomy or endometrial curettage on routine benign gynaecologic indications. Methods Endometrial tissue was obtained throughout the menstrual cycle. The presence of oxytocin mRNA was investigated by in situ hybridisation and by real time PCR. Main outcome measures Oxytocin mRNA signalling intensity found by in situ hybridisation of tissue obtained at different times of the menstrual cycle. Relative amounts of oxytocin mRNA measured by real time PCR. Results The signal for oxytocin mRNA obtained by in situ hybridisation was more pronounced in glandular epithelial cells than in stromal cells. Furthermore, it was most marked around mid-cycle. The expression of oxytocin mRNA was confirmed by real time PCR. Conclusions The results indicate that oxytocin may be synthesised in the endometrium of non-pregnant women, particularly in the glandular epithelial cells. Hormone released from these sources may have a paracrine action on the uterus. Oxytocin mRNA expression seems to be ovarian hormone dependent with the highest concentration around mid-cycle. [source] The role of arginine vasopressin in human labour: functional studies, fetal production and localisation of V1a receptor mRNABJOG : AN INTERNATIONAL JOURNAL OF OBSTETRICS & GYNAECOLOGY, Issue 1 2002S. Thornton Objective To investigate labour-associated changes in: 1. the myometrial contractile response to arginine vasopressin compared with oxytocin in vitro 2. fetal production of arginine vasopressin and 3. myometrial vasopressin V1a receptor mRNA. Design The contractile response to vasopressin (compared with oxytocin) was investigated in paired myometrial strips in vitro. Blood was taken from the umbilical artery and vein at delivery and arginine vasopressin measured by radio-immunoassay. V1a receptor mRNA was determined by in situ hybridisation. Results Myometrium was more sensitive to arginine vasopressin than oxytocin (P <0.05 for frequency, amplitude and activity integral in paired strips) after, but not before labour. There was a marked umbilical arteriovenous difference in arginine vasopressin concentration at delivery suggesting fetal production which was not influenced by labour. Myometrial vasopressin V1a receptor mRNA was not increased after the onset of labour. Conclusions The human uterus is extremely sensitive to arginine vasopressin in vitro. Arginine vasopressin is produced by the fetus but fetal formation is not increased during labour. [source] Two novel imatinib-responsive PDGFRA fusion genes in chronic eosinophilic leukaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2007Claire E. Curtis Summary We identified two patients with a t(2;4)(p24;q12) and a t(4;12)(q2?3;p1?2), respectively, in association with BCR-ABL and FIP1L1-PDGFRA negative chronic eosinophilic leukaemia. Molecular analysis revealed a novel STRN - PDGFRA fusion for the t(2;4) and ETV6 - PDGFRA for the t(4;12). The fusions were confirmed by specific amplification of the genomic breakpoints, reverse transcription polymerase chain reaction and fluorescence in situ hybridisation. Both patients were treated with imatinib and, following a rapid haematological response, achieved cytogenetic remission and a major molecular response. In conclusion, PDGFRA fuses to diverse partner genes in myeloid disorders. Identification of these fusions is important as they are particularly sensitive to imatinib. [source] | |||||||||||||||||||||||||