Down-regulation of caveolin-1, an inhibitor of transforming growth factor-beta signaling, in acute allergen-induced airway remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.     

NITPICK: peak identification for mass spectrometry data

Background  

The reliable extraction of features from mass spectra is a fundamental step in the automated analysis of proteomic mass spectrometry (MS) experiments.     

In vivo endothelial gene regulation in diabetes

Background

In comparison to the well established changes in compliance that occur at the large vessel level in diabetes, much less is known about the changes in compliance of the cardiovascular system at the end-organ level. The aim of this study was therefore to examine whether there was a correlation between resistance of the intrarenal arteries of the kidney and compliance of the left ventricle, as estimated by measurements of diastolic function, in subjects with type 2 diabetes.

Methods

We studied 167 unselected clinic patients with type 2 diabetes with a kidney duplex scan to estimate intrarenal vascular resistance, i.e. the resistance index (RI = peak systolic velocity-minimum diastolic velocity/peak systolic velocity) and a transthoracic echocardiogram (TTE) employing tissue doppler studies to document diastolic and systolic ventricular function.

Results

Renal RI was significantly higher in subjects with diastolic dysfunction (0.72 ± 0.05) when compared with those who had a normal TTE examination (0.66 ± 0.06, p < 0.01). Renal RI values were correlated with markers of diastolic dysfunction including the E/Vp ratio (r = 0.41, p < 0.001), left atrial area (r = 0.36, p < 0.001), the E/A ratio (r = 0.36, p < 0.001) and the E/E' ratio (r = 0.31, p < 0.001). These associations were independent of systolic function, hypertension, the presence and severity of chronic kidney disease, the use of renin-angiotensin inhibitors and other potentially confounding variables.

Conclusion

Increasing vascular resistance of the intrarenal arteries was associated with markers of diastolic dysfunction in subjects with type 2 diabetes. These findings are consistent with the hypothesis that vascular and cardiac stiffening in diabetes are manifestations of common pathophysiological mechanisms.     

PA,a stress-induced short cut to switch-on ethylene signalling by switching-off CTR1?

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.¹ PA was shown to inhibit (1) CTR1''s protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.Key words: lipid signaling, phosphatidic acid, ethylene, constitutive triple response 1, plant stress signaling, protein kinase, phospholipase D     

The utility of the morphological variation of pollen for resolving the evolutionary history of Billia (subfam. Hippocastanoideae,Sapindaceae)

In this study, we examined the utility of pollen morphology for resolving questions about the evolutionary history of Billia, which is a poorly known genus of Neotropical trees. Billia has been traditionally circumscribed with two species and treated as sister to Aesculus L. However, the number of species in Billia is uncertain, because the genus exhibits abundant morphological diversity but little discontinuous variation. Therefore, Billia may be monotypic and highly polymorphic, or it may have two species with blurred boundaries due to incipient speciation and/or hybridization. Moreover, one recent molecular phylogenetic study shows Billia nested withinAesculus. Our work sought to address the following questions: (i) Are there discontinuities in the pollen of Billia that may suggest species boundaries? (ii) Does the pollen of Billia show evidence for inter-specific hybridization? (iii) Do the exine morphology and size of pollen in Billia differ from those in Aesculus? Our results from scanning electron microscopy showed that pollen exine morphology is not taxonomically informative in Billia but that there are significant differences in pollen size between red- and white-flowered individuals. Thus, our pollen data support the utility of flower color in Billia for species delimitation. Our assessments of pollen viability do not support hybridization in the genus, but cannot be used to rule it out. Finally, pollen exine morphology may lend some support to an evolutionary origin ofBillia within eastern North American Aesculus. In contrast, data on pollen size suggest that Billia may belong in a topological position outside of Aesculus.     

Measurement and analysis of triplet-state lifetimes by multifrequency cross-correlation phase and modulation phosphorimetry.

In this paper we describe a novel approach to study the triplet-state lifetimes by a conventional multifrequency cross-correlation phase and modulation apparatus. The analysis of phase and modulation data of eosin-labeled band 3 erythrocyte ghosts revealed the existence of two phosphorescence lifetime values of 2700 and 750 microseconds, with a fractional contribution of 78 and 22%, respectively, which are in good agreement with those reported in the literature. Differential polarization phase analysis, which facilitates the study of the rotational properties of band 3, provided data in good agreement with those reported in the literature. The method proposed in this paper to study the radiative emission from the triplet state may represent a convenient alternative to the pulse laser flash technique.     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.