90 research outputs found

    Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study

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    BACKGROUND: Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer. METHODS: By Surface Enhanced Laser Desorption/Ionisation – Time Of Flight / Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells. RESULTS: Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells. CONCLUSIONS: HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression

    The volume activated potassium channel KCNK5 is up-regulated in activated human T Cells, but volume regulation is impaired

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    Background/Aims: The potential role of the two-pore domain potassium channel KCNK5 (also known as TASK-2 and K2P5.1) in activated T cell physiology has only recently been described. So far KCNK5 has been described to be up-regulated in T cells in multiple sclerosis patients and to be implicated in the volume regulatory mechanism regulatory volume decrease (RVD) in T cells. Methods: We investigated the time-dependent expression pattern of KCNK5 in CD3/CD28 activated human T cells using qPCR and Western blotting and its role in RVD using a Coulter Counter. Results: KCNK5 is highly up-regulated in CD3/CD28 activated T cells both at mRNA (after 24 h) and protein level (72 and 144 h), but despite this up-regulation the RVD response is inhibited. Furthermore, the swelling-activated Cl- permeability in activated T cells is strongly decreased, and the RVD inhibition is predominantly due to the decreased Cl- permeability. Conclusion: The up-regulated KCNK5 in activated human T cells does not play a volume regulatory role, due to decreased Cl- permeability. We speculate that the KCNK5 up-regulation might play a role in hyperpolarization of the cell membrane leading to increased Ca2+ influx and proliferation of T cells

    The Effect of Cataract Surgery on Circadian Photoentrainment A Randomized Trial of Blue-Blocking versus Neutral Intraocular Lenses

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    PurposeCataract decreases blue light transmission. Because of the selective blue light sensitivity of the retinal ganglion cells governing circadian photoentrainment, cataract may interfere with normal sleep–wake regulation and cause sleep disturbances. The purpose was to investigate the effect of cataract surgery on circadian photoentrainment and to determine any difference between blue-blocking and neutral intraocular lenses (IOLs).DesignThe study was a single-center, investigator-driven, double-masked, block-randomized clinical trial.ParticipantsOne eye in 76 patients with bilateral age-related cataract eligible for cataract surgery was included.MethodsIntervention was cataract surgery by phacoemulsification. Patients were randomized to receive a blue-blocking or neutral IOL.Main Outcome MeasuresPrimary outcome was activation of intrinsic photosensitive ganglion cells using post-illumination pupil response (PIPR) to blue light from 10 to 30 seconds after light exposure as a surrogate measure. Secondary outcomes were circadian rhythm analysis using actigraphy and 24-hour salivary melatonin measurements. Finally, objective and subjective sleep quality were determined by actigraphy and the Pittsburgh Sleep Quality Index.ResultsThe blue light PIPR increased 2 days (17%) and 3 weeks (24%) after surgery (P < 0.001). The majority of circadian and sleep-specific actigraphy parameters did not change after surgery. A forward shift of the circadian rhythm by 22 minutes (P = 0.004) for actigraphy and a tendency toward an earlier melatonin onset (P = 0.095) were found. Peak salivary melatonin concentration increased after surgery (P = 0.037). No difference was detected between blue-blocking and neutral IOLs, whereas low preoperative blue light transmission was inversely associated with an increase in PIPR (P = 0.021) and sleep efficiency (P = 0.048).ConclusionsCataract surgery increases photoreception by the photosensitive retinal ganglion cells. Because of inconsistency between the significant findings and the many parameters that were unchanged, we can conclude that cataract surgery does not adversely affect the circadian rhythm or sleep. Longer follow-up time and fellow eye surgery may reveal the significance of the subtle changes observed. We found no difference between blue-blocking and neutral IOLs, and, because of the minor effect of surgery in itself, an effect of IOL type seems highly unlikely

    Biased Signaling of the Angiotensin II Type 1 Receptor Can Be Mediated through Distinct Mechanisms

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    Seven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or β-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of β-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G protein-uncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit β-arrestins. Since uncoupling of G proteins by increased ability to recruit β-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms.We studied five AT1R mutants previously published to display pathway separation: D74N, DRY/AAY, Y292F, N298A, and Y302F (Ballesteros-Weinstein numbering: 2.50, 3.49-3.51, 7.43, 7.49, and 7.53). We find that D74N, DRY/AAY, and N298A mutants are more prone to β-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit β-arrestin in response to Sar1-Ile4-Ile8 (SII) Ang II, a ligand solely activating the β-arrestin pathway.Our analysis reveals that the underlying conformations induced by these AT1R mutants most likely represent principally different mechanisms of uncoupling the G protein, which for some mutants may be due to their increased ability to recruit β-arrestin2. Hereby, these findings have important implications for drug discovery and 7TMR biology and illustrate the necessity of uncovering the exact molecular determinants for G protein-coupling and β-arrestin recruitment, respectively

    Activation of p90(Rsk1) Is Sufficient for Differentiation of PC12 Cells

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    We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF. Rsk1 and Rsk2 mutated in the PDK1-binding site were not activated by either NGF or MEK1-DD. Expression of constitutively active Rsk1 or Rsk2 in PC12 cells resulted in highly active proteins whose levels of activity did not change either with NGF treatment or after coexpression with MEK1-DD. Rsk2-CA expression had no detectable effect on the cells. However, expression of Rsk1-CA led to differentiation of PC12 cells even in the absence of NGF, as evidenced by neurite outgrowth. Differentiation was not observed with a nonactive Rsk1-CA that was mutated in the PDK1-binding site. Expression of Rsk1-CA did not lead to activation of the endogenous MAPK pathway, indicating that Rsk1 is sufficient to induce neurite outgrowth and is the only target of MAPK required for this effect. Collectively, our data demonstrate a key role for Rsk1 in the differentiation process of PC12 cells
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