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Semiquantitative Reverse Transcription-polymerase Chain Reaction (semiquantitative + reverse_transcription-polymerase_chain_reaction)
Selected AbstractsEffects of metformin and oleic acid on adipocyte expression of resistinDIABETES OBESITY & METABOLISM, Issue 1 2006R Rea Aim:, The adipocyte-secreted hormone resistin has been implicated in obesity-induced insulin resistance and type 2 diabetes, but pharmacological and dietary factors that regulate resistin gene expression and the effects of resistin on cellular glucose uptake in muscle have not been clearly defined. Methods:, Expression of resistin mRNA was studied in differentiated 3T3-L1 adipocytes by using real-time semiquantitative reverse transcription-polymerase chain reaction. The effects of resistin on insulin-stimulated and insulin-independent 2-deoxyglucose uptake were evaluated in L6 muscle cells. Results:, Insulin 1 µm and rosiglitazone 10 µm markedly reduced resistin mRNA expression (relative to the control gene TF2D) by 4.7-fold (p < 0.05) and 5.3-fold (p < 0.02), respectively. Similar reductions in resistin mRNA were demonstrated with metformin 100 µm (6.2-fold reduction, p < 0.02) and oleic acid 100 µm (3.9-fold reduction, p < 0.03). Resistin 1 µm significantly reduced maximum insulin-stimulated 2-deoxyglucose uptake in L6 cells from 634 to 383% (relative to 100% for control, p < 0.001), and co-administration of rosiglitazone had no effect on resistin-induced insulin resistance. In the absence of insulin, however, resistin increased glucose uptake dose-dependently (e.g., 1.75-fold at 5 µm, p < 0.001) via a mitogen-activated protein kinase-dependent pathway. Conclusions:, These results demonstrate that various glucose-lowering therapies and oleic acid reduce resistin gene expression in isolated adipocytes, and that resistin impairs insulin-stimulated glucose uptake in skeletal muscle-derived cells. [source] Global analysis of gene expression profiles in ileum in a rat bladder augmentation model using cDNA microarraysINTERNATIONAL JOURNAL OF UROLOGY, Issue 11 2004HIDEAKI MIYAKE Abstract Background: The objective of the present study was to globally characterize the changes in the gene expression profile in the ileum after long-term urine exposure in a rat ileal augmented bladder model using cDNA microarrays. Methods: Bladder augmentation using the ileum was performed in female 8-week-old rats. The ileal epithelia used for bladder augmentation were harvested 1 and 3 months postoperatively and changes in the gene expression in these tissues were compared with that of intact ileal epithelia from sham-operated rats using cDNA microarrays consisting of 1176 rat genes. Results: Marked changes in gene expression in the ileum used for bladder augmentation were observed for 30 genes (16 up-regulated and 14 down-regulated genes). The differentially expressed genes include those associated with signal transduction, cell adhesion and stress response. Subsequent evaluation of changes in two randomly selected genes from the 30 differentially expressed genes by semiquantitative reverse transcription-polymerase chain reaction demonstrated the reliability of the present cDNA microarray analyses. Conclusion: The present experiments identified an extensive list of genes differentially expressed in the ileum after bladder augmentation, providing valuable information for the pathophysiological assessment of patients who undergo urinary reconstruction and representing a source of novel targets for treating complications after urinary diversion. [source] Alendronate Interacts With the Inhibitory Effect of 1,25(OH)2D3 on Parathyroid Hormone-Related Protein Expression In Human Osteoblastic Cells,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2003L Gómez-García Abstract The bisphosphonate alendronate is a potent inhibitor of bone resorption by its direct action on osteoclasts. In addition, there is some data suggesting that alendronate could also inhibit bone resorption indirectly by interacting with osteoblasts. Parathyroid hormone-related protein (PTHrP) produced by osteoblasts and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are regulators of bone remodeling, which have interrelated actions in these cells. In this study, we assessed whether alendronate can affect PTHrP expression in the presence or absence of 1,25(OH)2D3 in human primary osteoblastic (hOB) cells from trabecular bone. Cell total RNA was isolated, and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out using human PTHrP-specific primers. PTHrP in the hOB cell-conditioned medium was analyzed by a specific immunoradiometric assay. We found that PTHrP mRNA and secreted PTHrP were maximally inhibited by 10,8 -10,6 M of 1,25(OH)2D3 treatment within 8,72 h in hOB cells. Alendronate (10,14 -10,8 M) modified neither PTHrP mRNA nor PTHrP secretion, although it consistently abrogated the decrease in PTHrP production induced by 1,25(OH)2D3 in these cells. On the other hand, alendronate within the same dose range did not affect either the vitamin D receptor (VDR) mRNA or osteocalcin secretion, with or without 1,25(OH)2D3, in hOB cells. The inhibitory effect of alendronate on the 1,25(OH)2D3 -induced decrease in PTHrP in these cells was mimicked by the calcium ionophore A23187 (5 × 10,6 M), while it was eliminated by 5 × 10,5 M of nifedipine. Furthermore, although alendronate alone failed to affect [Ca2+]i in these cells, it stimulated [Ca2+]i after pretreatment of hOB cells with 10,8 M of 1,25(OH)2D3, an effect that was abolished by 5 × 10,5 M of nifedipine. These results show that alendronate disrupts the modulatory effect of 1,25(OH)2D3 on PTHrP production in hOB cells. Our findings indicate that an increase in calcium influx appears to be involved in the mechanism mediating this effect of alendronate. [source] Inhibitory effects of N -acetylcysteine on scavenger receptor class A expression in human macrophagesJOURNAL OF INTERNAL MEDICINE, Issue 5 2002L. SVENSSON Abstract.,Svensson L, Norén K, Wiklund O, Lindmark H, Ohlsson B, Mattsson Hultén L (Wallenberg Laboratory for Cardiovascular Research, The Sahlgrenska Academy at Göteborg University, Göteborg; and AstraZeneca, Mölndal, Sweden). Inhibitory effects of N -acetylcysteine on scavenger receptor class A expression in human macrophages. J Intern Med 2002; 251:. Objective.,The formation of foam cells from monocyte-derived macrophages involves the uptake of modified lipoproteins by scavenger receptors. Antioxidants inhibit lipoprotein oxidation and may also modulate gene expression. We investigated the effect of the antioxidant N -acetylcysteine on the expression of the class A scavenger receptor (SR-A) types I and II in human macrophages. Design.,Monocytes and macrophages from healthy blood donors and plaque-derived macrophages from patients undergoing carotid endartherectomy were used for experiments. SR-A mRNA was analysed with quantitative and semiquantitative reverse transcription-polymerase chain reaction, and ligand binding and uptake were assessed with 125I-labelled acetylated low-density lipoprotein (LDL). Results.,Incubation of monocytes and monocyte-derived macrophages with N -acetylcysteine decreased both SR-A I and II mRNA expression. N -Acetylcysteine also reduced SR-A mRNA in lesion-derived cells. Binding and uptake of 125I-acetylated LDL was decreased after brief incubation with N -acetylcysteine. After longer periods of incubation with N -acetylcysteine we observed an increased degradation of lipoproteins. Conclusions.,Our results imply that N -acetylcysteine leads to a decrease in SR-A mRNA and initially also to an attenuated uptake of modified lipoproteins. This adds more to the knowledge about the cellular actions of this drug. [source] Evidence for Increased Neuropeptide Y Synthesis in Mediobasal Hypothalamus in Relation to Parental Hyperphagia and Gonadal Activation in Breeding Ring DovesJOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2007S. Ramakrishnan Like lactating mammals, male and female ring dove parents increase their food consumption to meet the energetic challenges of provisioning their young. To clarify the neurochemical mechanisms involved, the present study investigated the relationship between parental hyperphagia and changes in activity of the potent orexigen neuropeptide Y (NPY) in the hypothalamus of breeding doves. Changes in NPY-immunoreactive (NPY-ir) cell numbers in the tuberal hypothalamus of male and female doves were examined by immunocytochemistry at six stages of the breeding cycle. Parallel NPY mRNA measurements were recorded in mediobasal hypothalamus (which includes the tuberal hypothalamus) by semiquantitative reverse transcription-polymerase chain reaction using 18S rRNA as the internal standard. NPY mRNA changes were also measured in the mediobasal hypothalamus of nonbreeding doves following intracranial administration of prolactin, an orexigenic hormone that is elevated in the plasma of parent doves, and in response to food deprivation, which mimics the negative energy state that develops in parents as they provision their growing young. NPY-ir cell numbers in the tuberal hypothalamus and NPY mRNA levels in the mediobasal hypothalamus were significantly higher in breeding males and females during the period of parental hyperphagia after hatching than during the late incubation period when food intake remains unchanged. In nonbreeding doves, food deprivation and prolactin treatment increased NPY mRNA in this region by two- to three-fold, which suggests that NPY expression is sensitive to hormonal and metabolic signals associated with parenting. We conclude that NPY synthesis is increased in the mediobasal hypothalamus during the posthatching period, which presumably supports increased NPY release and resulting parental hyperphagia. NPY-ir and mRNA were also high in the mediobasal hypothalamus prior to egg laying when food intake remained unchanged. Several lines of evidence suggest that this elevation in NPY supports the increased gonadal activity that accompanies intense courtship and nest building interactions in breeding doves. [source] Evidence for post-translational regulation of NrtA, the Aspergillus nidulans high-affinity nitrate transporterNEW PHYTOLOGIST, Issue 4 2007Ye Wang Summary ,,Here, influx and efflux of , and net fluxes of and , were measured in Aspergillus nidulans mutants niaD171 and niiA5, devoid of nitrate reductase (NR) and nitrite reductase (NiR) activities, respectively. ,,Transcript and protein abundances of NrtA, the A. nidulans principal high-affinity transporter, were determined using semiquantitative reverse transcription-polymerase chain reaction and western blots, respectively. , influx in niaD171 was negligible relative to wild-type values, whereas efflux to influx ratios increased nine-fold. Nevertheless, NrtA mRNA and NrtA protein were expressed at levels more than two-fold and three-fold higher, respectively, in niaD171 than in the wild-type strain. ,,This is the first demonstration of diminished high-affinity influx associated with elevated transporter levels, providing evidence that, in addition to transcriptional regulation, control of NrtA expression operates at the post-translational level. This mechanism allows for rapid control of transport at the protein level, reduces the extent of futile cycling of that would otherwise represent a significant energy drain when influx exceeds the capacity for assimilation or storage, and may be responsible for the rapid switching between the on and off state that is associated with simultaneous provision of to mycelia absorbing . [source] Adenosine receptor expression in Escherichia coli -infected and cytokine-stimulated human urinary tract epithelial cellsBJU INTERNATIONAL, Issue 11 2009Susanne Säve OBJECTIVE To assess the expression and regulation of adenosine receptors in unstimulated, uropathogenic Escherichia coli (UPEC)-infected and cytokine-stimulated human urinary tract epithelial cells, and to examine the regulation of interleukin (IL)-6 secretion in response to A2A receptor activation. MATERIALS AND METHODS Human urinary tract epithelial cells (A498, T24 and RT4) were grown in cell culture and stimulated with a mixture of pro-inflammatory cytokines (CM) or UPEC. The expression of adenosine receptors was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry. IL-6 secretion was measured with an enzyme-linked immunosorbent assay. RESULTS RT-PCR analysis showed the presence of transcripts for the A1, A2A and A2B receptor subtypes but not for the A3 receptor in A498 kidney epithelial cells. The expression of A2A receptor mRNA increased in A498 epithelial cells exposed to CM and UPEC, while A1 and A2B receptor transcripts decreased or remained unchanged. Up-regulation of A2A receptors was confirmed at the protein level using Western blot analysis and immunocytochemistry. There was also an increase in A2A receptor mRNA in human bladder epithelial cells (T24 and RT4) and in mouse bladder uroepithelium in response to cytokines and UPEC. IL-6 secretion in UPEC-infected A498 cells was decreased by 38% when exposed to the A2A receptor agonist CGS 21680. CONCLUSION Our data showed a subtype-selective plasticity among adenosine receptors in urinary tract epithelial cells in response to UPEC-infection and cytokines. There was a consistent up-regulation of A2A receptors in kidney and bladder epithelial cells. Functionally, A2A receptor activation reduced UPEC-induced IL-6 secretion. These findings suggest that adenosine might be a previously unrecognized regulator of the mucosal response in urinary tract infection. [source] | ||||||||||