Analytical potential of total reflection X-ray fluorescence spectrometry for simultaneous determination of iron, copper and zinc in human blood serum and plasma.

Due to many roles of trace elements such as Fe, Cu and Zn in various physiological and pathophysiological processes, their determination in serum and plasma is of high clinical relevance. In the present study, for the first time, the effect of serum and plasma preparation parameters (dilution factor and sample deposition volume) on the quality of results obtained by TXRF analysis was evaluated by means of experimental design tools (response surface analysis). It was found that the best strategy was the direct analysis of both human fluids without a previous dilution step. The accuracy and precision of the proposed methods were evaluated by analysis of reference materials (ClinChek® Plasma Control Level II and Seronorm™ Trace Elements Serum L-1). TXRF results agreed with the reference values and no significant differences at 95% confidence level were found. Limits of detection for the elements of interest were also adequate, taking into account their typical concentration ranges in real serum and plasma samples. Finally, the developed TXRF methods were applied to a set of serum and plasma samples from patients with different genders, ages and diagnoses, previously analysed by ICP-OES and ICP-MS techniques. The results showed good agreement between both analytical approaches. These results suggest that the proposed TXRF method provides reliable results thus being suitable for plasma and serum analysis, but in a simpler and more sustainable way.

Cloning and expression of cDNAs encoding alpha1,3-fucosyltransferase homologues from Arabidopsis thaliana.

The core alpha1,3-fucosyltransferases are involved in the synthesis of glycans specific to plants and invertebrates which are known to be immunogenic and allergenic. We report the identification, isolation and characterisation of the cDNAs of three genes (FucTA, FucTB and FucTC) encoding proteins similar to alpha1,3-fucosyltransferases in Arabidopsis thaliana. Reverse transcription-polymerase chain reaction was used to amplify the full length coding sequence of FucTA. The FucTA gene, which consists of seven exons, encodes a presumptive protein of 501 amino acids showing an overall sequence identity of 66% to the protein encoded by the recently isolated mung bean Fuc-T C3 cDNA. FucTA was expressed in Pichia pastoris under the control of the AOX1 gene promoter. The soluble enzyme was found to catalyse the same reaction as mung bean core alpha1,3-fucosyltransferase as judged by analyses of the products by MALDI-TOF and high-performance liquid chromatography. The FucTB cDNA was isolated from a lambda-ZAP library, but the clone used an alternative splicing site between the second and third exon resulting in a premature stop codon. The FucTC gene encodes a protein with less than 40% identity to FucTA across 115 amino acids of a total of 401 amino acids and is a member of a new sub-family of plant alpha1,3/4-fucosyltransferase homologues.

Galectin-3 decreases in mice exposed to immobilization stress.

Using M3/38 monoclonal antibody we have analyzed effects of immobilization stress on the expression of galectin-3 in liver, spleen and peritoneal macrophages from adult RFM and C3H mice, as well as from aged C3H mice (total of 22 animals). In all analyzed tissues, immobilization stress caused a significant decrease in the expression of galectin-3, ranging from 14% to 47%. The decrease of galectin-3 was observed in both strains of mice, as well as in old animals. Moreover, the same range of decrease (approximately 50%) was observed when cells grown in vitro were exposed to subculturing, or heat-shock. Although the biological significance of this effect is not known, it is very interesting that a single episode of immobilization stress was sufficient to cause a significant decrease in galectin-3, implicating that this beta-galactoside-binding lectin might be involved in the physiological response to psychological stress.

Expression of galectin-3 in cells exposed to stress-roles of jun and NF-kappaB.

BACKGROUND/AIMS: Galectin-3 is an interesting intracellular lectin that appears to be involved in numerous physiological processes. We have analyzed expression of galectin-3 in glioblastoma cells exposed to heat-shock, alkylating agents, UV-C radiation and subculturing (trypsinization). METHODS: Protein levels of galectin-3 were measured by western-blot analysis using M3/38 monoclonal antibody. The involvement of transcription factors NF-kappaB and Jun in the induction of galectin-3 was addressed using specific inhibitor of NF-kappaB (zL(3)-vs) and antisense-jun oligonucleotides. RESULTS: Exposure of cells to heat-shock or subculturing (trypsinization) decreased levels of galectin-3 to approximately 50%. Alkylating damage and UV-C irradiation caused an increase in the expression of galectin-3. Both inhibition of Jun by antisense-jun oligonucleotides, and inhibition of NF-kappaB by specific proteasomal inhibitor attenuated the induction of galectin-3 by UV-light, but with somewhat different kinetics. CONCLUSIONS: We have found that different forms of cellular stress have different effects on the expression of galectin-3. Heat-shock and subculturing decrease, while alkylating agents and UV-light increase galectin 3. NF-kappaB and Jun were shown to be involved in the induction of galectin-3 by UV-light, which is a first demonstration that these transcriptional factors are involved in the regulation of galectin-3 expression.

Transfer to in vitro conditions influences expression and intracellular distribution of galectin-3 in murine peritoneal macrophages.

Galectin-3 is a beta-galactoside-binding lectin that has been implicated in numerous physiological processes, including mRNA splicing, cell differentiation, tumor metastasis and the stress response. We have studied effects of transfer of resident murine peritoneal macrophages to in vitro conditions on galectin-3 in different cell compartments. Galectin-3 was purified by immunoprecipitation with rat monoclonal antibody M3/38, and analyzed by immunoblotting using the same antibody. Transfer to in vitro conditions nearly doubled the total amount of galectin-3 in cells, and caused significant alterations in its intracellular distribution, indicating that galectin-3 is involved in the adaptation of peritoneal macrophages to in vitro conditions.

Glycosylation of stress glycoprotein GP62 in cells exposed to heat-shock and subculturing.

GP62 is a member of the stress glycoprotein family that was proposed to have a chaperone-like function in the heat-shock response. Using lectin blotting we have studied glycosylation of GP62 and determined that in addition to heat-shock, even simple subculturing of cells is a sufficient stimulus to provoke induction of GP62. Interestingly, both kinetics of induction and glycosylation of GP62 induced by subculturing were different than when GP62 was induced by heat-shock. While GP62 induced by heat-shock was recognized by SNA, DSA and PHA-E lectins, and not by BSA I, Con A, RCA I, SJA, UEA I, VVA, and WGA lectins, GP62 induced by subculturing was also recognized by RCA I and WGA lectins.

Fucosylation of IgG heavy chains is increased in rheumatoid arthritis.

OBJECTIVES: Glycosylation of IgG was suggested to be important in the etiology of rheumatoid diseases. Most studies addressed the amount of galactose, but recently we showed that fucose is highly increased in the juvenile chronic arthritis. The objective of this study was to determine fucosylation of IgG heavy chains in patients with rheumatoid arthritis (RA). DESIGN AND METHODS: IgG was purified from sera of 29 RA patients and 17 matching controls using ammonium sulfate precipitation and ion exchange. Heavy chains were separated by denaturing polyacrylamide gel electrophoresis and their fucosylation analysed using fucose-specific UEA I lectin. RESULTS: Fucose was found to be approximately 40% increased in RA patients with very high statistical significance (p = 0.00095). CONCLUSIONS: Fucose on IgG heavy chains is significantly increased in patients with rheumatoid arthritis.

Stressin and natural killer cell activity in professional soldiers.

Chronic stress causes multiple biochemical and physiological changes in the human organism. Recently we have identified stressin, a human serum glycoprotein that was significantly increased in sera of prisoners released from Serbian concentration camps. To eliminate malnutrition and maltreatment as possible causes for the increased stressin concentration, we have analyzed stressin in sera of 40 professional soldiers after involvement in major military activity and compared it to stressin in sera of 20 control individuals. As expected, the sera of professional soldiers contained more than 2.2 times higher concentrations of stressin than control sera. It is interesting that, contrary to expectations, the natural killer cell activity of professional soldiers was normal or even increased. We hypothesize that this might be an effect of winning the war that could have, at least temporarily, erased the immunosuppressive effects of stress.

Changes of glycoprotein patterns in sera of humans under stress.

Stress exhibits adverse effects on many vital processes in which glycoproteins play a significant role(e.g. cell-cell/matrix interactions, immune response, neoplastic growth, implantation, prenatal development), yet only scarce attention has been directed towards studying stress induced changes in glycoprotein patterns. Using SDS-electrophoresis, blotting and digoxigenin-labelled lectins (Sambucus nigra agglutinin, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Maackia amurensis agglutinin and peanut (Arachis hypogaea) agglutinin),sera were analysed from 30 individuals chosen randomly from a severely stressed population of 309 male volunteers with no specific medical symptoms. Significant changes were found in glycoprotein pattern and content, compared with healthy controls of matching age and sex. Occasionally minor non-specific deviations from the reference values for several analytes (haemoglobin, glucose, bilirubin and alanine aminotransferase) were detected in the tested group, but glycoprotein GP4S (Mr = 45 000), detected by Datura stramonium agglutinin and Sambucus nigra agglutinin, appeared in 96.7% of samples of the stressed population. The same population also revealed an approximately 500-fold increase of GP37 in comparison with the control sera. These results suggest that stress, as a non-specific syndrome, induces specific biochemical changes, which could be of diagnostic relevance as risk makers before any more serious symptoms of stress-related consequences have developed.