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Confocal Microscopic Analysis (confocal + microscopic_analysis)
Selected AbstractsNoradrenergic 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] 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] Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytesBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2009M. Kobayashi Summary Background, Epidermal keratinocytes are involved in the skin innate immunity and express toll-like receptors (TLRs) and other innate immune proteins. The epidermis is continuously exposed to pathogenic Gram-positive bacteria or fungi. However, few studies have examined the function and expression of innate immune proteins in keratinocytes. Histamine, which is well known for itch and allergy, is closely associated with innate immunity, but its influence on epidermal innate immunity is still unclear. Objectives, To clarify the expression of innate immune proteins in keratinocytes stimulated by ligand pathogen-associated molecules, and the function of histamine in this process. Methods, We investigated the effects of lipopeptide (MALP-2, 1,100 ng mL,1; ligand for TLR2), peptidoglycan (PGN, 0·02,2 ,g mL,1; ligand for NOD2) and ,-glucan (1,100 ,g mL,1; ligand for dectin-1) in the presence or absence of histamine on mRNA expression of TLR2, NOD2 and dectin-1 as well as human ,-defensin 2 by quantitative real-time polymerase chain reaction in cultured normal human epidermal keratinocytes. TLR2 expression was also examined at the cell surface and intracellularly, as determined by flow cytometry and confocal microscopy. The quantities of interleukin (IL)-1, and IL-8 produced by keratinocytes were measured using enzyme-linked immunosorbent assay. Results, At the mRNA level, TLR2 was enhanced by PGN but not by its ligand MALP-2 or by ,-glucan; NOD2 was easily induced by all three ligands; and dectin-1 was enhanced by its ligand ,-glucan. These enhanced expressions were further augmented by histamine at 1 ,g mL,1. While the surface expression of TLR2 was barely detectable by flow cytometry even after stimulation, the intracellular expression of TLR2 was apparently elevated by PGN and further promoted by histamine. A confocal microscopic analysis also revealed the enhanced expression of TLR2 in the cytoplasm. The expression of TLR2, NOD2 and dectin-1 was functional, as these pathogen-associated molecules induced the production of IL-1,, IL-8 and defensin, and again, histamine greatly enhanced this production. Conclusions, Our study demonstrated that the expression of functional innate immune receptors is augmented by the pathogen-associated molecules in a ligand-feed forward or nonrelated manner in keratinocytes, and histamine promotes their expression and the resultant production of cytokines and defensins. [source] An in vivo confocal microscopic analysis of Salzmann's nodular degeneration: pre- and post-surgical interventionACTA OPHTHALMOLOGICA, Issue 2 2009Stephan Linke [source] | ||||||||||