Cannabinoid receptor types 1 and 2 and peroxisome proliferator-activated receptor-α: distribution in the skin of clinically healthy cats and cats with hypersensitivity dermatitis.

BACKGROUND: Cannabinoid receptors and peroxisome proliferator-activated receptor-alpha (PPAR-α) are gaining recognition as potential therapeutic targets for the treatment of skin disorders. HYPOTHESIS/OBJECTIVES: The aim of this study was to investigate the distribution of cannabinoid type 1 and 2 receptors (CBR1 and CBR2) and PPAR-α in feline skin and verify whether changes occur in the course of hypersensitivity dermatitis. ANIMALS: Twelve privately owned cats. Skin samples were obtained from five healthy cats with no skin lesions and seven cats clinically diagnosed with hypersensitivity dermatitis. METHODS AND MATERIALS: Haematoxylin and eosin stained skin sections were investigated for histopathological changes. Indirect immunofluorescence for CBR1, CBR2 and PPAR-α was performed on paraffin-embedded sections, and antibody specificity tested by Western blot analysis. RESULTS: Skin samples from cats with hypersensitivity dermatitis were all histopathologically diagnosed with eosinophilic dermatitis. CB receptors and PPAR-α were distributed throughout the skin in both healthy and allergic cats. In normal feline skin, these receptors were mainly distributed in the epithelial compartment. Receptor expression increased in hypersensitivity compared to healthy skin, with the main distribution changes being suprabasal for CBR1, dermal for CBR2 and marked expression of PPAR-α in hyperplastic epidermis and perivascular infiltrate. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased expression of cannabinoid receptors in the skin of cats with hypersensitivity dermatitis suggests an endogenous protective strategy and may support the use of natural cannabinoid receptor or PPAR-α agonists to treat feline hypersensitivity dermatitis.

TiO2 nanoparticles in seawater: Aggregation and interactions with the green alga Dunaliella tertiolecta.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely employed in industrial applications, thus rising concern about their impact in the aquatic environment. In this study we investigated the chemical behaviour of TiO2 NPs in the culture medium and its effect on the green alga Dunaliella tertiolecta, in terms of growth inhibition, oxidative stress, ROS (Reactive Oxygen Species) accumulation and chlorophyll content. In addition, the influence of exopolymeric substances (EPS) excreted by the microalgae on the stability of NPs has been evaluated. The physicochemical characterization showed a high propensity of TiO2 NPs to form micrometric-sized aggregates within 30min, large enough to partially settle to the bottom of the test vessel. Indeed, an increasing amount of TiO2 particles settled out with time, but the presence of EPS seemed to mitigate this behaviour in the first 6h of exposure where the main effects in D. tertiolecta were observed. TiO2 NPs did not inhibit the 72-h growth rate of D. tertiolecta, nor affected the cellular chlorophyll concentration in the range 0.01-10mgL-1. The time-course of ROS production showed an initial transient increase of ROS in TiO2 NP-exposed algae compared to the control, concomitant with an enhancement of catalase activity. Interestingly, intracellular ROS was a small fraction of total ROS, the highest amount being extracellular. The occurrence of cell-mediated chemical transformations of TiO2 NPs in the external medium, related to the presence of EPS, has been evaluated. Our results showed that carbohydrates were the major component of EPS, whereas proteins of medium molecular weight (20-80kDa) were preferentially bound to TiO2 NPs, likely influencing their biological fate.

High-Flux Dialysis: Clinical, Biochemical, and Proteomic Comparison with Low-Flux Dialysis and On-Line Hemodiafiltration.

Hemodiafiltration on-line (on-line HDF) is a more efficient treatment than low-flux hemodialysis (HD). Unfortunately, it cannot be proposed to all patients. The aim of this study was to evaluate the safety, efficiency, and mechanisms of removal of toxins with high-flux HD vs. low-flux HD and on-line HDF. Randomized cross-over study designed to evaluate efficiency and tolerability of high-flux HD vs. low-flux HD in aged patients; to compare by means of biochemical and proteomic analyses the efficiency and mechanisms of removal of toxins with high-flux HD vs. on-line HDF. The removal of small toxins was similar with high-flux and low-flux HD. ß2-microglobulin was removed only with high-flux HD, which had an excellent tolerability. The efficiency of high-flux HD was similar to on-line HDF. Proteomic analysis demonstrated that only high-flux membranes remove and adsorb small proteins. High-flux HD may be an efficient alternative to on-line HDF.

ß-trace protein is highly removed during haemodialysis with high-flux and super high-flux membranes.

BACKGROUND: Serum ß-trace protein (ßTP, MW 23-29 kDa) is a marker of GFR impairment in renal patients. Recent papers propose to predict residual renal function (RRF) in maintenance haemodialysis (MHD) patients from serum concentrations of ßTP and other small proteins, avoiding the collection of urine. Few data are available on the removal of ßTP in patients treated with dialysis membranes with different flux characteristics. The aim of this study was to evaluate the effects of haemodialysis with low-flux, high-flux and super high-flux membranes on serum concentrations of ßTP in MHD patients with null RRF. METHODS: Serum ßTP concentrations were measured before and after the first dialysis of the week in 51 MDH patients treated by low-flux (n = 24), high-flux (n = 17), or super high-flux (n = 10) membranes. The removal of ß2-microglobulin (ß2M, MW 11.8), cystatin C (Cys, MW 13.3), urea and creatinine was also analyzed. RESULTS: Low-flux membranes did not remove ßTP, ß2M and Cys whose concentration increased at the end of dialysis. High-flux membrane removed more efficiently ß2M and Cys than ßTP. Super high-flux membrane had the highest efficiency to remove ßTP: mean reduction ratio (RR) 53.4%, similar to ß2M (59.5%), and Cys (62.0%). CONCLUSIONS: In conclusion, the plasma clearance of small proteins and particularly of ßTP is dependent from the permeability of the dialysis membranes Therefore, the reliability of the formulas proposed to predict RRF from serum ßTP and other LMWP may be affected by the different permeability of the dialysis membranes.

A SELDI-TOF approach to ecotoxicology: comparative profiling of low molecular weight proteins from a marine diatom exposed to CdSe/ZnS quantum dots.

Quantum dots (QDs), namely semiconductor nanocrystals, due to their particular optical and electronic properties, have growing applications in device technology, biotechnology and biomedical fields. Nevertheless, the possible threat to human health and the environment have attracted increasing attention as the production and applications of QDs increases rapidly while standard evaluation of safety lags. In the present study we performed proteomic analyses, by means of 2D gel electrophoresis and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS). We aimed to identify potential biomarkers of exposure to CdSe/ZnS quantum dots. The marine diatom Phaeodactylum tricornutum exposed to 2.5nM QDs was used as a model system. Both 2DE and SELDI showed the presence of differentially expressed proteins. By Principal Component Analysis (PCA) we were able to show that the differentially expressed proteins can discriminate between exposed and not exposed cells. Furthermore, a protein profile specific for exposed cells was obtained by SELDI analysis. To our knowledge, this is the first example of the application of SELDI technology to the analysis of microorganisms used as biological sentinel model of marine environmental pollution.

The response of Phaeodactylum tricornutum to quantum dot exposure: Acclimation and changes in protein expression.

Nanotechnology has a great potential to improve life and environmental quality, however the fate of nanomaterials in the ecosystems, their bioavailability and potential toxicity on living organisms are still largely unknown, mainly in the marine environment. Genomics and proteomics are powerful tools for understanding molecular mechanisms triggered by nanoparticle exposure. In this work we investigated the effect of exposure to CdSe/ZnS quantum dots (QDs) in the marine diatom Phaeodactylum tricornutum, using different physiological, biochemical and molecular approaches. The results show that acclimation to QDs reduced the growth inhibition induced by nanoparticles in P. tricornutum cultures. The increase of glutathione observed at the end of the lag phase pointed to cellular stress. Transcriptional expression of selected stress responsive genes showed up-regulation in the QD-exposed algae. A comparison of the proteomes of exposed and unexposed cells highlighted a large number of differentially expressed proteins. To our knowledge, this is the first report on proteome analysis of a marine microalga exposed to nanoparticles.

Inhibition of metalloproteinase activity in FANCA is linked to altered oxygen metabolism.

Bone marrow (BM) failure, increased risk of myelodysplastic syndrome, acute leukaemia and solid tumors, endocrinopathies and congenital abnormalities are the major clinical problems in Fanconi anemia patients (FA). Chromosome instability and DNA repair defects are the cellular characteristics used for the clinical diagnosis. However, these biological defects are not sufficient to explain all the clinical phenotype of FA patients. The known defects are structural alteration in cell cytoskeleton, altered structural organization for intermediate filaments, nuclear lamina, and mitochondria. These are associated with different expression and/or maturation of the structural proteins vimentin, mitofilin, and lamin A/C suggesting the involvement of metalloproteinases (MPs). Matrix metalloproteinases (MMP) are involved in normal physiological processes such as human skeletal tissue development, maturation, and hematopoietic reconstitution after bone marrow suppression. Current observations upon the eventual role of MPs in FA cells are largely inconclusive. We evaluated the overall MPs activity in FA complementation group A (FANCA) cells by exposing them to the antioxidants N-acetyl cysteine (NAC) and resveratrol (RV). This work supports the hypothesis that treatment of Fanconi patients with antioxidants may be important in FA therapy.

Reappraisal of quantitative gel zymography for matrix metalloproteinases.

BACKGROUND: The determination of matrix metalloproteases (MMPs) is relevant in many pathophysiological conditions, especially if associated with extracellular matrix remodeling; however, the results obtained are closely linked to the method used and are not directly comparable. The aim of this study was to perform a reappraisal of quantitative gel zymography technique for MMPs in human plasma, to use for comparison with commercially available ELISA and in those experimental conditions where the MMP active form needs to be revealed. METHODS: The critical methodological parameters of zymography were checked and a comparison with a routinely used ELISA was performed. RESULTS: Sensitivity and reproducibility levels of zymography are suitable for detection of MMP-9 in human plasma, providing results closely related to those obtained by ELISA. CONCLUSIONS: Analytical parameters of zymography were suitable for detection of MMPs in human plasma. Quantitative zymography for MMPs is an alternative method for comparing the results of ELISA widely employed for MMP determination, thus reducing the discrepancies between laboratories regarding gelatinase assay.

Temperature and pressure dependence of azurin stability as monitored by tryptophan fluorescence and phosphorescence. The case of F29A mutant.

The effects of a single-point, F29A, cavity-forming mutation on the unfolding thermodynamic parameters of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift in the fluorescence spectrum, led to a volume change of 70-90mlmol(-1). The difference in the unfolding volume between F29A and wild type azurin was smaller than the volume of the space theoretically created in the mutant, indicating that the cavity is, at least partially, filled with water molecules. The complex temperature dependence of the unfolding volume, for temperatures up to 20°C, suggests the formation of an expanded form of the protein and highlights how the packing efficiency of azurin appears to contribute to the magnitude of internal void volume at any given temperature. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime. At 40°C the application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between wild type and the cavity mutant is the inversion point which happens at 300MPa for wild type and at 150MPa for F29A. This suggests that, for the cavity mutant, pressure-induced internal hydration is more dominant than any compaction of the globular fold at relatively low pressures.

Prediction of glomerular filtration rate from serum concentration of cystatin C: comparison of two analytical methods.

BACKGROUND: The aim of this study was to compare two methods used to measure serum cystatin C (Cys) and their accuracy to predict glomerular filtration rate (GFR). METHODS: Three hundred and sixty-seven adult chronic kidney disease (CKD) patients with different functional impairments participated in this study. GFR was determined as the renal clearance of 99mTc-DTPA. Serum concentrations of cystatin C (SCys) were determined with an immunonephelometric method and with an immunoturbidimetric method. RESULTS: A very high linear correlation was found between the two measurements of SCys (r=0.929). The mean difference of SCysTurb-SCysNeph was 0.02±0.43 mg/L (not significant). A high logarithmic correlation was also found between SCys and GFR (r was 0.919 for SCysNeph and 0.937 for SCysTurb). By means of multiple regression analysis, we developed formulae to predict GFR from SCysNeph, SCysTurb and SCr. For comparison, GFR was predicted using published formulae. A good agreement was found between predicted GFR and measured GFR. The results showed that the accuracy of SCysNeph, SCysTurb and SCr and of the different prediction formulae were quite similar. CONCLUSIONS: The immunoturbidimetric method seems adequate to measure SCys and to predict GFR and its impairment in CKD, at least similar to the immunonephelometric method. The accuracy of SCys and of derived formulae was not higher than that of SCr and SCr-based formulae.

Albumin modification and fragmentation in renal disease.

Albumin is the most important antioxidant substance in plasma and performs many physiological functions. Furthermore, albumin is the major carrier of endogenous molecules and exogenous ligands. This paper reviews the importance of post-translational modifications of albumin and fragments thereof in patients with renal disease. First, current views and controversies on renal handling of proteins, mainly albumin, will be discussed. Post-translational modifications, namely the fragmentation of albumin found with proteomic techniques in nephrotic patients, diabetics, and ESRD patients will be presented and discussed. It is reasonable to hypothesize that proteolytic fragmentation of serum albumin is due to a higher susceptibility to proteases, induced by oxidative stress. The clinical relevance of the fragmentation of albumin has not yet been established. These modifications could affect some physiological functions of albumin and have a patho-physiological role in uremic syndrome. Proteomic analysis of serum allows the identification of over-expressed proteins and can detect post-translational modifications of serum proteins, hitherto hidden, using standard laboratory techniques.