Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%–19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.     

Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni

Abstract A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni . Amplimers of the FlaA gene obtained by PCR were digested with Alu I and Hin fI to distinguish C. coli from C. jejuni . With Alu I digestion C. jejuni -specific bands were observed at 110, 140 and 160 bp and C. coli -specific bands at 293 and 147 bp. C. jejuni -specific bands of 349 and 109 bp were found by Hin fI digestion but Hin fI did not digest the Fla A amplimer of C. coli . This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.     

High Mobility Group 1 Protein Is Not Stably Associated with the Chromosomes of Somatic Cells

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.     

Low-dose dopamine as an activator of renal catecholamine receptors of different classes. I. Studies in water-salt depletion

The renal function in healthy man with salt and water depletion induced by natriuretic treatment was explored during steady hypotonic polyuria. Four 15 min clearance (cl.) periods, before, during and after dopamine (DA) infusion in a subpressor dose were performed. The 12 examined subjects showed different renal hemodynamic responses in the early stage of DA infusion, i.e. hyperemic (4 subjects, subgroup A) or ischemic (8 subjects, subgroup B). A decrease in urinary sodium excretion and increase in tubular sodium reabsorption, in particular at the diluting segment level, were induced by DA in both subgroups, at least in the late stage of infusion. During the control cl. period in subgroup A as compared with B the renal plasma flow was lower and the tubular sodium reabsorption higher, suggesting a relatively higher level of renal adrenergic activity.     

Na+-ATPase in the gills of bass (Dicentrarchus labrax)

The authors evidence a Mg2+ dependent ATPase activity stimulated by Na+ in absence of K+ in bass gill microsomes. As this stimulated ATPase shows different features from "baseline" activity measured in the absence of both Na+ and K+ ions (Mg2+-ATPase) and from 1mM ouabain sensitive (Na+ + K+)-ATPase, it has been ascribed to a distinct Na+-ATPase. In the present paper the optimal conditions for bass gill Na+-ATPase assay and the temperature dependence of the enzyme are reported. Moreover the Na+-ATPase appears to be insensitive to 1mM ouabain and 100% inhibited by 2,5mM ethacrynic acid. It is suggested a parallel diffusion of Na+- and (Na+ + K+)-ATPase and a possible physiological role of Na+ATPase in osmoregulation.     

Anti-thirst action of propranolol

A reduction of the thirst was observed one hour after intramuscolar injection of 0.3 mg/kg propranolol in the rat; this effect was not observed with higher doses. According to work hypotheses, small doses could act blocking renal beta-adrenergic receptors: the stopped renin emission reduces angiotensin production that is the basic factor of thirst. The AA hypothesize that the lack of the effect in response to higher doses of propranolol can be explained through a different action of this drug which antagonizes the first one.     

Variations in plasma protein components during extracorporeal circulation

In this research we have determined the behaviour of proteic plasmatic pattern and some enzymes during extracorporeal circulation and we noted a constant increase of albumin and of the ratio Alb/Glob. We observed also variations of some enzymes. Our opinion according other AA. is that this changes are determined principally by the large dose of heparin necessary during the C.E.C.     

Separation of mannosylretinylphosphate from dolichylmannosylphosphate by chromatography on columns of DEAE-sephacel

A one-step column chromatographic procedure on DEAE-Sephacel allows the separation of mannosylretinylphosphate from dolichylmannosylphosphate with minimal breakdown of the mannosylretinylphosphate. Using this procedure, subcellular fractions of rat liver were shown to be active in synthesizing both mannolipids from GDP-[¹⁴C]mannose in the absence or presence of exogenous retinylphosphate.