The soluble proteome of tobacco Bright Yellow-2 cells undergoing H₂O₂-induced programmed cell death

Plant programmed cell death (PCD) is a genetically controlled process that plays an important role in development and stress responses. Reactive oxygen species (ROS) are key inducers of PCD. The addition of 50 mM H?O? to tobacco Bright Yellow-2 (TBY-2) cell cultures induces PCD. A comparative proteomic analysis of TBY-2 cells treated with 50 mM H?O? for 30 min and 3 h was performed. The results showed early down-regulation of several elements in the cellular redox hub and inhibition of the protein repair-degradation system. The expression patterns of proteins involved in the homeostatic response, in particular those associated with metabolism, were consistently altered. The changes in abundance of several cytoskeleton proteins confirmed the active role of the cytoskeleton in PCD signalling. Cells undergoing H?O?-induced PCD fail to cope with oxidative stress. The antioxidant defence system and the anti-PCD signalling cascades are inhibited. This promotes a genetically programmed cell suicide pathway. Fifteen differentially expressed proteins showed an expression pattern similar to that previously observed in TBY-2 cells undergoing heat shock-induced PCD. The possibility that these proteins are part of a core complex required for PCD induction is discussed.     

Drug releasing systems in cardiovascular tissue engineering

Heart disease and atherosclerosis are the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts; however, current artificial grafts carry significant limitations, including thrombosis, infection, limited durability and the inability to grow. Tissue engineering of blood vessels, cardiovascular structures and whole organs is a promising approach for creating replacement tissues to repair congenital defects and/or diseased tissues. In an attempt to surmount the shortcomings of artificial grafts, tissue-engineered cardiovascular graft (TECVG), constructs obtained using cultured autologous vascular cells seeded onto a synthetic biodegradable polymer scaffold, have been developed. Autologous TECVGs have the potential advantages of growth, durability, resistance to infection, and freedom from problems of rejection, thrombogenicity and donor scarcity. Moreover polymers engrafted with growth factors, cytokines, drugs have been developed allowing drug-releasing systems capable of focused and localized delivery of molecules depending on the environmental requirements and the milieu in which the scaffold is placed. A broad range of applications for compound-releasing, tissue-engineered grafts have been suggested ranging from drug delivery to gene therapy. This review will describe advances in the development of drug-delivery systems for cardiovascular applications focusing on the manufacturing techniques and on the compounds delivered by these systems to date.     

Molecular cloning and biochemical characterization of the skin tyrosinase from Rana esculenta L.

Amphibian tyrosinases display unique and poorly understood properties such as seasonal activity variations, different activities in dorsal and ventral skin and the occurrence as inactive forms requiring proteolytic activation. For the first time we have sequenced and characterized Rana esculenta L. tyrosinase by functional expression of the cloned cDNA, and compared it with frog skin extracts. R. esculenta tyrosinase ORF is well conserved compared with tyrosinases of various sources. The amino acid similarities between the tyrosinases from R. esculenta and other amphibia range from 85% to 98%. Homology remains high with mammalian tyrosinases (65% identity with Homo sapiens, and 63% with Mus musculus) and with bird orthologues (66% identity with Gallus gallus). Tyrosinase was expressed in HEK293T cells as an active enzyme. Activity staining on non reducing SDS-PAGE revealed two bands around 63 and 68 kDa. R. esculenta skin extracts were mildly active and reached maximal activity upon protease treatment, revealing a high molecular weight dopa-positive band in the 200 kDa range and one of higher MW, after nagarse treatment, in activity stainings. The different behaviour of recombinant tyrosinase compared to skin extracts suggests formation in vivo of a multimeric complex.     

GITR signaling potentiates airway hyperresponsiveness by enhancing Th2 cell activity in a mouse model of asthma

Background

Anxiety and depression are common and treatable risk factors for re-hospitalisation and death in patients with COPD. The degree of lung function impairment does not sufficiently explain anxiety and depression. The BODE index allows a functional classification of COPD beyond FEV₁. The aim of this cross-sectional study was (1) to test whether the BODE index is superior to the GOLD classification for explaining anxious and depressive symptoms; and (2) to assess which components of the BODE index are associated with these psychological aspects of COPD.

Methods

COPD was classified according to the GOLD stages based on FEV_1%predicted in 122 stable patients with COPD. An additional four stage classification was constructed based on the quartiles of the BODE index. The hospital anxiety and depression scale was used to assess anxious and depressive symptoms.

Results

The overall prevalence of anxious and depressive symptoms was 49% and 52%, respectively. The prevalence of anxious symptoms increased with increasing BODE stages but not with increasing GOLD stages. The prevalence of depressive symptoms increased with both increasing GOLD and BODE stages. The BODE index was superior to FEV_1%predicted for explaining anxious and depressive symptoms. Anxious symptoms were explained by dyspnoea. Depressive symptoms were explained by both dyspnoea and reduced exercise capacity.

Conclusion

The BODE index is superior to the GOLD classification for explaining anxious and depressive symptoms in COPD patients. These psychological consequences of the disease may play a role in future classification systems of COPD.     

Characterization of denatured proteins by hydrophobic interaction chromatography: a preliminary study

In this preliminary study hydrophobic interaction chromatography (HIC) is proposed as a good tool in order to detect conformational changes induced by chemical denaturants in two globular proteins, cytochrome C (Cyt C) and myoglobin (MYO). Alterations in protein structure were manifested chromatographically by reproducible changes in peak heights, retention time, and appearance of multiple peaks. The HIC behavior of the two model proteins denatured by guanidinium thyocyanate (GdmSCN) was investigated, keeping constant various concentrations of urea in the mobile phase in a TSK-Gel Phenyl-5PW column (TosoBiosep). Suitable elution conditions provide evidence of the simultaneous presence of two denatured forms in the case of MYO, and sequential different denatured states of Cyt C.     

Cyclic uridine diphosphate glucose: a new pyrimidine analog of cyclic ADP ribose

Novel compound 1, as the first example of cyclic ADP-ribose analogs containing a pyrimidine residue, was synthesized by a chemical strategy employing a Mitsunobu reaction for the condensation of the glucosyl moiety on protected uridine, and a Matsuda procedure for the cyclization step.     

Monitoring filamentous bulking in activated sludge systems fed by synthetic or municipal wastewater

The stability with respect to filamentous bulking of two activated sludge fully-aerobic systems, one with a completely mixed tank and one with a channel reactor, fed either by a synthetic wastewater or by a primary-settled municipal wastewater, of variable composition and flow rate, has been investigated. The morphological characteristics of the biomass in terms of floc size and roughness and of filamentous bacteria abundance have been monitored by image analysis. Severe bulking was only observed in the well-mixed tank fed at a constant flow rate by synthetic substrate of constant concentration, when the channel reactor fed in a similar manner was fully stable. Variations of biomass characteristics as well as of settling properties were observed on both systems fed with the real wastewater, but these events were related to the characteristics of the wastewater, as similar changes were observed on the full-scale plant fed with the same substrate. In any case, automated image analysis was an efficient way to monitor in detail the fate of the activated sludge at pilot and full scale.