Analysis of the oxido-redox status of plasma proteins. Technology advances for clinical applications

Reactive oxygen species (ROS) are potentially implicated in renal pathology. Direct evidence is available for animal models of glomerulonephritis but the demonstration of ROS implication in human diseases is only circumstantial and requires further experimental support. One problem limiting any evolution is the brief life of ROS (in terms of milliseconds) that makes it difficult their direct detection 'in vivo'. An alternative is to look at the products of oxidation of proteins that remain in blood as a signature of ROS activity. Recent data have shown the presence of oxidation products of albumin (sulfonic (34)Cys albumin) in serum of patients with focal-glomerulosclerosis, that is a primary glomerular diseases causing nephrotic syndrome. Structural studies based on spectroscopy and calorimetry strengthened the relevance of oxidation of the unique free SH groups of (34)Cys for conformation of albumin, in analogy with what already reported for other proteins. In this review, we present new developments on technologies for the detection of the oxido-redox potential of proteins that are based on the concept that oxidation is inversely correlated with their free content of sulphydryl groups. We describe, in particular, two new iodoacetamide-substituted cyanines that have been developed for labelling sulphydryl groups and can be utilized as stable dyes prior mono- and bi-dimensional electrophoresis. Proteins with low binding with iodoacetamide-cyanines may be considered as surrogate biomarkers of ROS activity. Standardization of these techniques and their acquisition in more laboratories would enable clinicians to plan screening studies on ROS in human diseases.     

Extensive netting in Albasini and Nandoni dams: a potential threat to fish as a sustainable food source in the Vhembe District,South Africa

Fish populations in Albasini and Nandoni dams are negatively affected by extensive netting practices. This observation was made by the authors following a number of fish health assessment surveys related to aquatic pollution in the Luvuvhu River catchment. A comparison between the number and size of fish collected over a period of ten years indicated decreases in the average size and a consistent low number of fish, despite similar extensive sampling efforts. Unregulated netting is a common practice in both dams. This may become a serious problem as fish from these two dams are an important source of protein for the local communities. The purpose of this note is to report that gillnets are illegally used in the system and on the effect this could have on the fish population. The authors suggest educational and awareness initiatives to inform local communities about the importance of utilising fish in a sustainable manner to ensure the livelihood of generations to come.     

Is disturbed clearance of apoptotic keratinocytes responsible for UVB-induced inflammatory skin lesions in systemic lupus erythematosus?

Apoptotic cells are thought to play an essential role in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesise that delayed or altered clearance of apoptotic cells after UV irradiation will lead to inflammation in the skin of SLE patients. Fifteen SLE patients and 13 controls were irradiated with two minimal erythemal doses (MEDs) of ultraviolet B light (UVB). Subsequently, skin biopsies were analysed (immuno)histologically, over 10 days, for numbers of apoptotic cells, T cells, macrophages, and deposition of immunoglobulin and complement. Additionally, to compare results with cutaneous lesions of SLE patients, 20 biopsies of lupus erythematosus (LE) skin lesions were analysed morphologically for apoptotic cells and infiltrate. Clearance rate of apoptotic cells after irradiation did not differ between patients and controls. Influx of macrophages in dermal and epidermal layers was significantly increased in patients compared with controls. Five out of 15 patients developed a dermal infiltrate that was associated with increased epidermal influx of T cells and macrophages but not with numbers of apoptotic cells or epidermal deposition of immunoglobulins. Macrophages were ingesting multiple apoptotic bodies. Inflammatory lesions in these patients were localised near accumulations of apoptotic keratinocytes similar as was seen in the majority of LE skin lesions. In vivo clearance rate of apoptotic cells is comparable between SLE patients and controls. However, the presence of inflammatory lesions in the vicinity of apoptotic cells, as observed both in UVB-induced and in LE skin lesions in SLE patients, suggests that these lesions result from an inflammatory clearance of apoptotic cells.     

Long-term experimental evolution in Escherichia coli. XI. Rejection of non-transitive interactions as cause of declining rate of adaptation

Background  

Experimental populations of Escherichia coli have evolved for 20,000 generations in a uniform environment. Their rate of improvement, as measured in competitions with the ancestor in that environment, has declined substantially over this period. This deceleration has been interpreted as the bacteria approaching a peak or plateau in a fitness landscape. Alternatively, this deceleration might be caused by non-transitive competitive interactions, in particular such that the measured advantage of later genotypes relative to earlier ones would be greater if they competed directly.     

Computational annotation of genes differentially expressed along olive fruit development

Background  

Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software.     

New iodo‐acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido‐redox status

Two new iodoacetamide‐substituted cyanines, C3NIASO3 and C5NIASO3, were synthesized starting from hemicyanine and were utilized for labeling plasma proteins. Specificity, sensitivity and feasibility for SH residues was tested utilizing an equimolar mixture of standard proteins and with normal plasma. Oxidized plasma proteins following H₂O₂ exposure and plasma from patients with focal glomerulosclerosis were analyzed as models of altered protein oxido‐redox status. Following optimization of the assay (dye/protein ratio, pH), C3NIASO3 and C5NIASO3 gave a sensitivity slightly better than N‐hydroxysuccinimidyl dyes for plasma proteins and were successfully employed for differential display electrophoresis (DIGE). Twenty‐nine proteins were detected in normal plasma after 2‐DE while less proteins were detected in plasma of patients with glomerulosclerosis. Following massive ‘in vitro' oxidation with H₂O₂, C3NIASO3 and C5NIASO3 failed to detect any residual SH, implicating massive oxidation. In conclusion, this study describes the synthesis of two new iodoacetamide cyanines that can be utilized for the analysis of plasma proteins with 2‐DE and DIGE. They are also indicated for the definition of the oxido‐redox status of proteins and were successfully utilized to extend the analysis of oxidation damage in patients with glomerulosclerosis.     

2D-electrophoresis and the urine proteome map: Where do we stand?

The discovery of urinary biomarkers is a main topic in clinical medicine. The development of proteomics has rapidly changed the knowledge on urine protein composition and probably will modify it again. Two-dimensional electrophoresis (2D-PAGE) coupled with mass spectrometry has represented for years the technique of choice for the analysis of urine proteins and it is time to draw some conclusions.This review will focus on major methodological aspects related to urine sample collection, storage and analysis by 2D-PAGE and attempt to define an advanced normal urine protein map.Overall, 1118 spots were reproducibly found in normal urine samples but only 275 were characterized as isoforms of 82 proteins. One-hundred height spots belonging to 30 proteins were also detected in plasma and corresponded to typical plasma components. The identity of most of the proteins found in normal urine by 2D-PAGE remains to be determined, the majority being low-molecular weight proteins (< 30 kDa). Equalization procedures would also enhance sensitivity of the analysis and allow low abundance proteins to be characterized.Therefore, we are still on the way to define the normal urine composition. Technology advancements in concentrating procedure will improve sensitivity and give the possibility to purify proteins for mass spectrometry.     

The PINE study: rationale and design of a randomised comparison of epidural injection of local anaesthetics and steroids versus care-as-usual to prevent postherpetic neuralgia in the elderly [ISRCTN32866390]

BACKGROUND: Postherpetic neuralgia (PHN) is by far the most common complication of herpes zoster (HZ) and one of the most intractable pain disorders. Since PHN is seen most often in the elderly, the number of patients with this disorder is expected to increase in our ageing society. PHN may last for months to years and has a high impact on the quality of life. The results of PHN treatment are rather disappointing. Epidural injection of local anaesthetics and steroids in the acute phase of HZ is a promising therapy for the prevention of PHN. Since randomised trials on the effectiveness of this intervention are lacking, the PINE (Prevention by epidural Injection of postherpetic Neuralgia in the Elderly) study was set up. The PINE study compares the effectiveness and cost-effectiveness of a single epidural injection of local anaesthetics and steroids during the acute phase of HZ with that of care-as-usual (i.e. antivirals and analgesics) in preventing PHN in elderly patients. METHODS / DESIGN: The PINE study is an open, multicenter clinical trial in which 550 elderly (age >/= 50 yr.) patients who consult their general practitioner in the acute phase of HZ (rash < 7 days) are randomised to one of the treatment groups. The primary clinical endpoint is the presence of HZ-related pain one month after the onset of the rash. Secondary endpoints include duration and severity of pain, re-interventions aiming to treat the existing pain, side effects, quality of life, and cost-effectiveness. CONCLUSION: The PINE study is aimed to quantify the (cost-) effectiveness of a single epidural injection during the acute phase of HZ on the prevention of PHN.     

Reaction of lysine with aldoses

The reactivity of amino acids with trioses and hexoses under physiological conditions (37°, pH 7.5) has been investigated. The most reactive species were lysine, glyceraldehyde, and 2-amino-2-deoxyglucose. They generated yellow, oligomeric compounds having molecular weights in the range 500–1000 and pI values in the range pH 4–6. In the Lys-glyceraldehyde adduct, the ε-amino group is incorporated into a pyrrole-type structure, which gives a positive reaction with the Pauly reagent. The largest oligomers correspond to the condensation of 4 mol of Lys with 8–10 mol of glyceraldehyde. It is hypothesised that the primary Amadori-compound rearranges during the Maillard reaction to give browning products, since no yellow oligomers are produced if the Lys-aldehyde adduct is reduced immediately with cyanoborohydride.