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Jasminder Sahi Michael P. Wiggins Gil B. Gibori Thomas J. Layden Mrinalini C. Rao 《Journal of cellular physiology》1996,168(2):276-283
To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit CI− secretion, their effects on CI− transport and intracellular Ca2+ concentrations ([Ca2+]i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl− sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca2+-sensitive fluorescent probe, fura-2AM were used to assess Cl− transport and [Ca2+]i, respectively. Basal Cl− transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl− channel blocker diphenylamine-2-carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na+/K+/2Cl− cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I− > F− ≥ Cl− > Br−. DPC inhibited I− influx by ∼50%, F− influx by 80%, Cl− influx by 85%, and Br− influx by 90%. Furosemide significantly inhibited influx of Br− (84%) and Cl− (81%) but not of F− and I−. The effects of agents known to alter biological response by increasing [Ca2+]i in other epithelial systems were used to stimulate Cl− transport. Cl− influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca2+ ionophore A23187 (2.05 ± 0.40). The Cl− permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl− influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl− permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl− transport by activating the Na+/K+/2Cl− cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl− channels and the cotransporter, and their actions are cyclooxygenase-independent. © 1996 Wiley-Liss, Inc. 相似文献
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A pulsed (17 nanoseconds) Nd:YAG laser (1064 nm) was used to inject impermeable dyes (propidium iodide andiodide and merocyanine 540) and a plasmid (pEGFP-N1) encoding green fluorescent protein (GFP) into human breast adenocarcinoma cells (MCF-7). The cell membrane integrity and viability were fully preserved in this laser-assisted transfer. 相似文献
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Gundeep Kaur Supreet Singh Harpreet Singh Mrinalini Chawla Tanima Dutta Harsimran Kaur Kyle Bender W. A. Snedden Sanjay Kapoor Ashwani Pareek Prabhjeet Singh 《PloS one》2015,10(8)
Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu2+, which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca2+-dependent, and CaM-binding domain is localized to 35–70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca2+ signaling and cyclophilin activity in Arabidopsis. 相似文献
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Nang Thu Thu Kyaw Anthony D. Harries Palanivel Chinnakali Annick Antierens Kyi Pyar Soe Mike Woodman Mrinalini Das Sharmila Shetty Moe Khine Lwin Zuu Pyae Sone Htwe Marcelo Fernandez 《PloS one》2015,10(8)
Background
Since 2004, Médecins Sans Frontières-Switzerland has provided treatment and care for people living with HIV in Dawei, Myanmar. Renal function is routinely monitored in patients on tenofovir (TDF)-based antiretroviral treatment (ART), and this provides an opportunity to measure incidence and risk factors for renal dysfunction.Methods
We used routinely collected program data on all patients aged ≥15 years starting first-line TDF-based ART between January 2012 and December 2013. Creatinine clearance (CrCl) was assessed at base line and six-monthly, with renal dysfunction defined as CrCl < 50ml/min/1.73m2. We calculated incidence of renal dysfunction and used Cox regression analysis to identify associated risk factors.Results
There were 1391 patients, of whom 1372 had normal renal function at baseline. Of these, 86 (6.3%) developed renal dysfunction during a median time of follow-up 1.14 years with an incidence rate of 5.4 per 100 person-years: 78 had CrCl between 30–50ml/min/1.73m2 and were maintained on TDF–based ART, but 5 were changed to another regimen: 4 because of CrCl <30ml/min/1.73m2. Risk factors for renal dysfunction included age ≥45 years, diagnosed diabetes, underlying renal disease, underweight and CD4 count <200cells/mm3. There were 19 patients with baseline renal dysfunction and all continued on TDF-based ART: CrCl stayed between 30–49 ml/min/1.73m2 in five patients while the remainder regained normal renal function.Conclusions
In a resource-poor country like Myanmar, the low incidence of renal toxicity in our patient cohort suggests that routine assessment of CrCl may not be needed and could be targeted to high risk groups if resources permit. 相似文献7.
I Bruce M Akhlaq GC Bloomfield E Budd B Cox B Cuenoud P Finan P Gedeck J Hatto JF Hayler D Head T Keller L Kirman C Leblanc DL Grand C McCarthy D O'Connor C Owen MS Oza G Pilgrim NE Press L Sviridenko L Whitehead 《Bioorganic & medicinal chemistry letters》2012,22(17):5445-5450
Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK. 相似文献
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Martinho O Granja S Jaraquemada T Caeiro C Miranda-Gonçalves V Honavar M Costa P Damasceno M Rosner MR Lopes JM Reis RM 《PloS one》2012,7(1):e30769
Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear. In the present study, we found that RKIP protein is absent in a low frequency (10%, 20/193) of glioma tumors. Nevertheless, the absence of RKIP expression was an independent prognostic marker in glioma. Additionally, by in vitro downregulation of RKIP, we found that RKIP inhibition induces a higher viability and migration of the cells, having no effect on cellular proliferation and angiogenesis, as assessed by in vivo CAM assay. In conclusion, this is the largest series studied so far evaluating the expression levels of this important cancer suppressor protein in glioma tumors. Our results suggest that in a subset of tumors, the absence of RKIP associates with highly malignant behavior and poor survival of patients, which may be a useful biomarker for tailored treatment of glioma patients. 相似文献
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Wang J Nikrad MP Travanty EA Zhou B Phang T Gao B Alford T Ito Y Nahreini P Hartshorn K Wentworth D Dinarello CA Mason RJ 《PloS one》2012,7(3):e29879
Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1β activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo. 相似文献
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