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Hybridization Signal (hybridization + signal)
Selected AbstractsKaryotype analysis and physical mapping of 45S rRNA genes in Hydrangea species by fluorescence in situ hybridizationPLANT BREEDING, Issue 3 2008K. Van Laere Abstract Detailed karyotypes of Hydrangea macrophylla, Hydrangea paniculata and Hydrangea quercifolia were constructed on the basis of arm lengths and centromeric index, together with 45S rDNA fluorescence in situ hybridization. Although the chromosomes were small, they were well distinguishable for all species. Chromosome morphology and karyotypes were different for the three species. H. macrophylla had six metacentric (M), eight submetacentric (SM) and four subtelocentric (ST) chromosomes. The karyotype of H. paniculata contained seven M, 10 SM and one ST chromosomes and H. quercifolia had six M, 10 SM and two ST chromosomes. The variability among three species also was expressed by 45S rDNA signals. H. macrophylla had a nucleolar organizing region on chromosome 2, H. paniculata had 45S rDNA signals on chromosomes 2, 5 and 11 and H. quercifolia on chromosomes 3 and 8. Hybridization signal always was distally on the short arm but the strength of the signals was different for the three species. The chromosome portraits made in this study will be used to trace chromosome behaviour in interspecific hybrids resulting from breeding work between the three species. [source] Cell type- and region-specific expression of protein kinase C-substrate mRNAs in the cerebellum of the macaque monkeyTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2003Noriyuki Higo Abstract We performed nonradioactive in situ hybridization histochemistry in the monkey cerebellum to investigate the localization of protein kinase C-substrate (growth-associated protein-43 [GAP-43], myristoylated alanine-rich C-kinase substrate [MARCKS], and neurogranin) mRNAs. Hybridization signals for GAP-43 mRNA were observed in the molecular and granule cell layers of both infant and adult cerebellar cortices. Signals for MARCKS mRNA were observed in the molecular, Purkinje cell, and granule cell layers of both infant and adult cortices. Moreover, both GAP-43 and MARCKS mRNAs were expressed in the external granule cell layer of the infant cortex. In the adult cerebellar vermis, signals for both GAP-43 and MARCKS mRNAs were more intense in lobules I, IX, and X than in the remaining lobules. In the adult hemisphere, both mRNAs were more intense in the flocculus and the dorsal paraflocculus than in other lobules. Such lobule-specific expressions were not prominent in the infant cerebellar cortex. Signals for neurogranin, a postsynaptic substrate for protein kinase C, were weak or not detectable in any regions of either the infant or adult cerebellar cortex. The prominent signals for MARCKS mRNA were observed in the deep cerebellar nuclei, but signals for both GAP-43 and neurogranin mRNAs were weak or not detectable. The prominent signals for both GAP-43 and MARCKS mRNAs were observed in the inferior olive, but signals for neurogranin were weak or not detectable. The cell type- and region-specific expression of GAP-43 and MARCKS mRNAs in the cerebellum may be related to functional specialization regarding plasticity in each type of cell and each region of the cerebellum. J. Comp. Neurol. 467:135,149, 2003. © 2003 Wiley-Liss, Inc. [source] Dynamics of 17,-Ethynylestradiol exposure in rainbow trout (Oncorhynchus mykiss): Absorption, tissue distribution, and hepatic gene expression patternENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 11 2006Ann D. Skillman Abstract 17,-Ethynylestradiol (EE2) is a synthetic estrogen identified in sewage effluents. To understand better the absorption kinetics of EE2 and the induction of vitellogenin (VTG) and estrogen receptor , (ER,) mRNA, we subjected male rainbow trout (Onchorynchus mykiss) to continuous water exposures of 125 ng/L of EE2 for up to 61 d. Trout were either repetitively sampled for blood plasma or serially killed at selected time intervals. Vitellogenin, ER, mRNA, and EE2 were measured using enzymelinked immunosorbent assay and using quantitative polymerase chain reaction and gas chromatography,mass spectrometry, respectively. In separate experiments, trout were exposed to EE2 for 7 d, and hepatic gene expression was assessed using a low- and high-density cDNA microarray. The EE2 was rapidly absorbed by the trout, with an apparent equilibrium at 16 h in plasma and liver. The ER, mRNA levels also increased rapidly, reaching near-peak levels by 48 h. In contrast, plasma levels of VTG continuously increased for 19 d. After 61 d, tissues with the highest levels of VTG were the liver, kidney, and testes. Microarray-based gene expression studies provided unexpected results. In some cases, known estrogen-responsive genes (e.g., ER,) were unresponsive, whereas many of the genes that have no apparent link to estrogen function or EE2 toxicity were significantly altered in expression. Of the two microarray approaches tested in the present study, the high-density array appeared to be superior because of the improved quality of the hybridization signal and the robustness of the response in terms of the number of genes identified as being EE2 responsive. [source] Cloning and Characterization of a Novel Purple Acid Phosphatase Gene (MtPAP1) from Medicago truncatula Barrel MedicJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 2 2006Kai Xiao Abstract A novel purple acid phosphatase gene (MtPAP1) was isolated from the model legume Medicago truncatula Barrel Medic. The cDNA was 1 698 bp in length with an open reading frame (ORF) of 1 398 bp capable of encoding an N-terminal signal peptide of 23 amino acids. The transcripts of MtPAP1 were mainly detected in leaves under high-phosphate conditions, whereas under low-phosphate conditions the transcript level was reduced in leaves and increased in roots, with the strongest hybridization signal detected in roots. A chimeric gene construct fusing MtPAP1 and GFP was made in which the fusion was driven by the CaMV35S promoter. Transgenic Arabidopsis plants carrying the chimeric gene constructs showed that the fusion protein was mainly located at the apoplast based on confocal microscopic analysis, showing that MtPAP1 could be secreted to the outside of the cell directed by the signal peptide at the N-terminal. The coding region of MtPAP1 without signal peptide was inserted into the prokaryotic expression vector pET-30a (+) and overexpressed in Escherichia coli BL21 (DE3). The acid phosphatase (APase) proteins extracted from bacterial culture were found largely based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An enzyme activity assay demonstrated that the APase activity in the transformed bacteria was 3.16-fold higher than that of control. The results imply that MtPAP1 functions to improve phosphorus acquisition in plants under conditions of phosphorus (P) stress. (Managing editor: Li-Hui Zhao) [source] Sensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot HybridizationJOURNAL OF PHYTOPATHOLOGY, Issue 6 2006L. Barbarossa Abstract A non-radioactive dot-blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)-labelled minus-sense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection. [source] Detection of Water Buffalo Sex Chromosomes in Spermatozoa by Fluorescence in situ HybridizationREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2003T Révay Contents In order to identify X- and Y-bearing spermatozoa in water buffalo by fluorescence in situ hybridization (FISH), some available probes of closely related species were examined. An X- and Y-specific probe set, made from flow sorted yak chromosomes, labelled in somatic metaphases of water buffalo the whole X and Y, respectively, except their centromere regions. A cattle Y-chromosome repeat sequence (BC1.2) showed strong signal on the telomere region of the buffalo Y-chromosome, demonstrating the evolutionary conservation of this locus in water buffalo. In hybridization experiments with spermatozoa from five buffaloes, the yak X-Y paint set demonstrated clear signals in more than 92% (46.8% X and 45.8% Y) of the cells. Using the cattle Y-chromosome specific BC1.2 probe, clear hybridization signal was detected in more than 48% of the cells. Statistical analysis showed that there was no significant difference between bulls or from the expected 50 : 50 ratio of X- and Y-bearing cells. The probes presented here are reliable to assess separation of X- and Y-bearing spermatozoa. [source] Granulocyte/macrophage colony-stimulating factor treatment of human chronic ulcers promotes angiogenesis associated with de novo vascular endothelial growth factor transcription in the ulcer bedBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2006F. Cianfarani Summary Background, Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine with pleiotropic functions, has been successfully employed in the treatment of chronic skin ulcers. The biological effects underlying GM-CSF action in impaired wound healing have been only partly clarified. Objectives, To investigate the effects of GM-CSF treatment of chronic venous ulcers on lesion vascularization and on the local synthesis of the angiogenic factors vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). Methods, Patients with nonhealing venous leg ulcers were treated with intradermal injection of recombinant human GM-CSF, and biopsies were taken at the ulcer margin before and 5 days after administration. Wound vascularization was analysed by immunohistochemistry using antiplatelet endothelial cell adhesion molecule-1/CD31 and anti-,-smooth muscle actin antibodies. VEGF and PlGF transcription was assessed by in situ hybridization. To identify the cell populations transcribing VEGF within the ulcer bed, the VEGF hybridization signal was correlated with the immunostaining for different cell type markers on serial sections. Direct induction of VEGF transcription by GM-CSF was investigated in GM-CSF-treated cultured macrophages and keratinocytes. Results, Blood vessel density was significantly increased in the ulcer bed following GM-CSF treatment. VEGF transcripts were localized in keratinocytes at the ulcer margin both before and after GM-CSF treatment, whereas a VEGF hybridization signal was evident within the ulcer bed only following administration. PlGF mRNA was barely detectable in keratinocytes at the ulcer margin and was not visibly increased after treatment. Unlike VEGF, a specific PlGF hybridization signal could not be detected in cells within the ulcer following GM-CSF administration. Monocytes/macrophages were the main cell population transcribing VEGF after GM-CSF treatment. In vitro analysis demonstrated that VEGF transcription can be directly stimulated by GM-CSF in a differentiated monocytic cell line, but not in keratinocytes. Conclusions, Our data show that increased vascularization is associated with GM-CSF treatment of chronic venous ulcers and indicate that inflammatory cell-derived VEGF may act as an angiogenic mediator of the healing effect of GM-CSF in chronic ulcers. [source] Demonstration of hydroxyindole-O-methyltransferase (HIOMT) mRNA expression in pineal parenchymal tumors: Histochemical in situ hybridizationJOURNAL OF PINEAL RESEARCH, Issue 4 2000Itaru Tsumanuma The expression of hydroxyindole-O-methyltransferase (HIOMT), an enzyme catalyzing the final step of melatonin biosynthesis, was examined in three pineoblastomas and five pineocytomas by in situ hybridization analysis. Distinct hybridization signals for HIOMT mRNA, though weaker than in normal pineal gland pinealocytes, were detected in two of the three pineoblastoma and three of the five pineocytoma cases. Of the pineoblastomas, hybridization signals were observed in most tumor cells of one case, while in another, signals were detected in occasional cells clustered or scattered throughout the neoplastic field. Of the pineocytomas, signals were detected in most tumor cells of two cases, while in one case, signals were detected only in occasional cells. Among these specimens, one pineoblastoma and one pineocytoma were also analyzed using northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses. In the northern blot analysis, an apparently single band corresponding to the size of HIOMT mRNA was detected in both pineoblastoma and pineocytoma RNA blots. In the RT-PCR analysis, three species of HIOMT mRNA generated via alternative splicing were detected in both tumors. These results suggest that the neoplastic cells of pineoblastomas and pineocytomas often retain the ability to express HIOMT mRNA, as in normal pinealocytes, and that HIOMT is a useful tumor marker for the diagnosis of pineal parenchymal tumors. [source] IgA nephropathy and mesangial cell proliferation: shared global gene expression profilesNEPHROLOGY, Issue 2002Hideto SAKAI SUMMARY: It is well established that mesangial cell proliferation plays a major role in glomerular injury and progressive renal injury. the expression of a number of different genes has been reported in proliferative mesangial cells in culture. However, the relevance of these genes to renal injury in general and IgA nephropathy (IgAN) remains to be established. Assessment of gene activity on a global genome-wide scale is a fundamental and newly developed molecular strategy to expand the scope of clinical investigation from a single gene to studying all genes at once in a systematic pattern. Capitalizing on the recently developed methodology of high cDNA array hybridization, the simultaneous expression of thousands of genes in primary human proliferating mesangial cells was monitored and compared with renal tissue of IgAN. Complex [,- 33P]-labelled cDNA targets were prepared from cultured mesangial cells, remnant tissue from five IgAN renal biopsies and four nephrectomies (controls). Each target was hybridized to a high-density array of 18 326 paired target genes. the radioactive hybridization signals were analysed by phosphorimager. Approximately 8212±530 different gene transcripts were detected per target. Close to 5% (386±90 genes) were full-length mRNA human transcripts (HT) and the remainder were expressed sequence tags (EST). Using a relational database, electronic subtraction was performed and matching was carried out to allow identification of 203 HT with shared expression in proliferative mesangial cells and IgAN renal biopsies. In addition hierarchical clustering analysis was performed on the HT of IgAN and controls to establish differential expression profiles of mesangial HT in IgAN and controls. Collectively the presented data constitutes a preliminary renal bioinformatics database of the transcriptional profiles in IgAN. More importantly, the information may help to speed up the discovery of genes underlying human IgAN. [source] Chromosome topology in normal and aneuploid blastomeres from human embryosPRENATAL DIAGNOSIS, Issue 12 2007Jan Diblík Abstract Objectives To find whether chromosomes 13, 16, 18, 21, 22, X and Y in blastomeres of human embryos are nonrandomly localized, whether their aneuploidy affects their localization and if eventual early inactivation of chromosome X with peripheral localization is present. Methods Relative distances from the nucleus center and edge of 1198 fluorescence in situ hybridization signals in 98 human blastomeres were measured in digital images for comparison with a mathematical model of random distribution in spherical nucleus. Results Comparison with the mathematical model revealed that localization of chromosomes 13, 16, 21, 22, X and Y in normal and aneuploid blastomeres and that of chromosome 18 in normal blastomeres was not significantly different from random distribution. Similarly, chromosome X in blastomeres with more than one X did not appear to have a preferential localization. Only chromosome 18 in aneuploid blastomeres was differently distributed (p < 0.0001) with a shift to the nuclear periphery (p = < 0.0001). Conclusions Peripheral localization of chromosome 18 in aneuploid blastomeres is related to embryo aneuploidy. Conversely, a peripheral localization of the inactive X chromosome was not found in blastomeres from 3-4 day old embryos. These results open the possibility to improve embryo selection after pre-implantation diagnosis. Copyright © 2007 John Wiley & Sons, Ltd. [source] Inhibition of adenylyl cyclase by neuronal P2Y receptorsBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2002Ursula Unterberger P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y12. Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment. ADP and ATP (0.1 , 100 ,M) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A2A or recombinant ,2 receptors. Forskolin-dependent cyclic AMP production was reduced by 1 ,M and enhanced by 10 , 100 ,M ADP; this latter effect was turned into an inhibition when A2A receptors were blocked. The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin. The rank order of agonist potencies for the reduction of cyclic AMP was (IC50 values): 2-methylthio-ADP (0.12 nM)=2-methylthio-ATP (0.13 nM)>ADP,S (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2,,4,-disulphonic acid, or adenosine-3,-phosphate-5,-phosphate, but attenuated by reactive blue 2, ATP,S, and 2-methylthio-AMP. RT , PCR demonstrated the expression of P2Y2, P2Y4, P2Y6, and P2Y12, but not P2Y1, receptors in PC12 cells. In Northern blots, only P2Y2 and P2Y12 were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged. We conclude that P2Y12 receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity. British Journal of Pharmacology (2002) 135, 673,684; doi:10.1038/sj.bjp.0704514 [source] | |||||||||||||