Gergely Montskó

Use of fullerene‐, octadecyl‐, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen

SPE plays a crucial role in bioanalytical research. In the present work a novel fullerene(C60)-derivatised silica material is compared with octadecyl(C18) - and triaconthyl(C30)-silicas regarding recoveries of peptides and sequence coverage of HSA and fibrinogen digests. C30- and C60(30 nm)-SPE materials were found to be the two most prominent SPE materials. At low peptide concentrations C60-material prepared from a silica gel with a pore size of 30 nm has proven to be the best material with regards to recoveries. By increasing the amount of loaded peptides recoveries decrease due to its relative low binding capacity in contrast to C30-silica particles, showing no changes. The best sequence coverages of Aalpha- and Bbeta-chains of 20 pmol fibrinogen digest can also be achieved using these two SPE materials, C60 (30 nm) demonstrates an outstanding value of sequence coverage (62.15%) achieved for the gamma-chain. After nonenzymatic glycation the digests of fibrinogen and HSA were also separated. This makes the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. For HSA, ten new sites of glycation at lysine and arginine residues have been explored. Using the detailed SPE/off-line MALDI method the glycation sites on fibrinogen are first described in this paper.

Flavonoid diosmetin increases ATP levels in kidney cells and relieves ATP depleting effect of ochratoxin A

Diosmetin (DIOS) is a flavone aglycone commonly occurring in citrus species and olive leaves, in addition it is one of the active ingredients of some medications. Based on both in vitro and in vivo studies several beneficial effects are attributed to DIOS but the biochemical background of its action seems to be complex and it has not been completely explored yet. Previous investigations suggest that most of the flavonoid aglycones have negative effect on ATP synthesis in a dose dependent manner. In our study 17 flavonoids were tested and interestingly DIOS caused a significant elevation of intracellular ATP levels after 6- and 12-h incubation in MDCK kidney cells. In order to understand the mechanism of action, intracellular ATP and protein levels, ATP/ADP ratio, cell viability and ROS levels were determined after DIOS treatment. In addition, impacts of different enzyme inhibitors and effect of DIOS on isolated rat liver mitochondria were also tested. Finally, the influence of DIOS on the ATP depleting effect of the mycotoxin, ochratoxin A was also investigated. Our major conclusions are the followings: DIOS increases intracellular ATP levels both in kidney and in liver cells. Inhibition of glycolysis or citric acid cycle does not decrease the observed effect. DIOS-induced elevation of ATP levels is completely abolished by the inhibition of ATP synthase. DIOS is able to completely reverse the ATP-depleting effect of the mycotoxin, ochratoxin A. Most probably the DIOS-induced impact on ATP system does not originate from the antioxidant property of DIOS. Based on our findings DIOS may be promising agent to positively influence ATP depletion caused by some metabolic poisons.

Analysis of nonderivatized steroids by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using C70 fullerene as matrix

Neutral steroid hormones are currently analyzed by gas or liquid chromatography/mass spectrometry based methods. Most of the steroid compounds, however, lack volatility and do not contain polar groups, which results in inadequate chromatographic behavior and low ionization efficiency. Derivatization of the steroids to form more volatile, thermostable, and charged products solves this difficulty, but the derivatization of compounds with unknown chemical moieties is not an easy task. In this study, a rapid, high-throughput, sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method is described using C70 fullerene as a matrix compound. The application of the method is demonstrated for five general sex steroids and for synthetic steroid compounds in both negative and positive ionization modes.

Lithium Induces ER Stress and N-Glycan Modification in Galactose-Grown Jurkat Cells

We previously reported that lithium had a significant impact on Ca2+ regulation and induced unfolded protein response (UPR) in yeast cells grown on galactose due to inhibition of phosphoglucomutase (PGM), however the exact mechanism has not been established yet. In this study, we analysed lithiums effect in galactose-fed cells to clarify whether these ER-related changes are the result of a relative hypoglycemic state. Furthermore, we investigated whether the alterations in galactose metabolism impact protein post-translational modifications. Thus, Jurkat cells were incubated in glucose or galactose containing media with or without lithium treatment. We found that galactose-fed and lithium treated cells showed better survivability than fasting cells. We also found higher UDP-Hexose and glycogen levels in these cells compared to fasting cells. On the other hand, the UPR (X-box binding protein 1 mRNA levels) of galactose-fed and lithium treated cells was even greater than in fasting cells. We also found increased amount of proteins that contained N-linked N-acetyl-glucosamine, similar to what was reported in fasting cells by a recent study. Our results demonstrate that lithium treatment of galactose-fed cells can induce stress responses similar to hypoglycemia, however cell survival is still secured by alternative pathways. We propose that clarifying this process might be an important addition toward the better understanding of the molecular mechanisms that regulate ER-associated stress response.

Induction of mitochondrial destabilization and necrotic cell death by apolar mitochondria-directed SOD mimetics.

In this paper, we present evidence, for the first time, that increasing the lipophilicity of mitochondria targeting SOD mimetics reverses their cytoprotective properties, destabilizing the mitochondrial membrane system and promoting cell death. A new mitochondria-directed apolar SOD mimetic, HO-3814, was found to provoke mitochondrial swelling and loss of mitochondrial membrane potential, and these effects were not inhibited by cyclosporine A. HO-3814-induced cell death was predominantly necrotic, caspase-independent, and not affected by mitochondrial permeability transition inhibitors or cyclophilin D-suppression, inhibitors of mitogen-activated protein kinases or Akt, or various antioxidants. In contrast, Bcl-2 overexpression diminished the effects of HO-3814.

Free and total cortisol levels are useful prognostic markers in critically ill patients: a prospective observational study

OBJECTIVE The role of cortisol in the prediction of mortality risk in critical illness is controversial in the literature. The aim of this study was to evaluate the prognostic value of cortisol concentrations in a mixed population of critically ill patients in medical emergencies. DESIGN In this prospective, observational study, measurement of total (TC) and free cortisol (FC) levels was made in the serum samples of 69 critically ill patients (39 males and 30 females, median age of 74 years) at admission (0 h) and 6, 24, 48, and 96 h after admission. METHODS Cortisol levels were determined using HPLC coupled high-resolution ESI-TOF mass spectrometry. The severity of disease was calculated by prognostic scores. Statistical analyses were performed using the SPSS 22.0 software. RESULTS The range of TC varied between 49.9 and 8797.8 nmol/l, FC between 0.4 and 759.9 nmol/l. The levels of FC at 0, 6, 24, and 48 h and TC at 0, 6 h were significantly elevated in non-survivors and correlated with the predicted mortality. The prognostic value of these cortisol levels was comparable with the routinely used mortality scores. In predictive models, FC at 6, 24, and 48 h proved to be an independent determinant of mortality. CONCLUSIONS The predictive values of FC in the first 2 days after admission and TC within 6 h are comparable with the complex, routinely used mortality scores in evaluating the prognosis of critically ill patients. The cortisol response probably reflects the severity of disease.

Alpha-1 chain of human haptoglobin as viability marker of in vitro fertilized human embryos: information beyond morphology

ABSTRACT Only one third of the in vitro fertilization treatments result in successful delivery following morphological viability assessment worldwide. A paper by Montskó et al. (2015) describes the identification of the alpha-1 chain of human haptoglobin as a potential marker of embryo viability. Using mass spectrometry, the concentration of the haptoglobin alpha-1 chain was determined in spent culture media samples of in vitro fertilized embryos and correlation was found with the outcome of the respective transfer. In the present study we investigated, whether the concentration of haptoglobin alpha-1 chain shows any correlation with morphological scores to clarify whether levels of the alpha-1 chain provide additional information on embryo viability unnoticed by the morphological assessment. In the study, pregnancy and live birth rates were examined in 143 transferred samples of 86 patients, retrospectively. Two sample groups were created. The control group contained embryos classified as ‘good’ or ‘fair’ based on the Istanbul Consensus Criteria System, while the double-assay group contained embryos assessed as ‘good’ or ‘fair’ by the morphological evaluation and as ‘viable’ by the haptoglobin assay. Clinical pregnancy rate was 30.2% in the control group, while 47.6% in the group scored parallel with morphological criteria and proteomic analysis (p < 0.05). The increased clinical pregnancy rate observed in the double-assayed group can be attributed to decreased false-positivity of the double assay. Abbreviations: IVF: in vitro fertilization; SEC: spent embryo culture medium; HSA: human serum albumin; Hpt: haptoglobin; HptA1: haptoglobin alpha-1 chain; ICCS: Istanbul Consensus Criteria System; BMI: body mass index; ICSI: intracytoplasmic sperm injection