Design, synthesis and properties of novel powerful antioxidants, glutathione analogues

Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC(50) of GSH: 1,231.0 +/- 311.8 microM; EC(50) of UPF peptides: from 0.03 to 35 microM) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC(50): 11.4 +/- 1.0 microM). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 microM concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.     

An antioxidant probiotic reduces postprandial lipemia and oxidative stress

Reducing postprandial oxidative stress (OxS), decreasing postprandial blood triglyceride level (TG) and improving lipoprotein status is likely to have a preventive impact on the development of cardiovascular disease (CVD). Previously we have shown that the antioxidant probiotic Lactobacillus fermentum ME-3 (DSM14241) is characterized by antiatherogenic effects. This randomized double-blind placebo-controlled study evaluated the influence of kefir enriched with an antioxidative probiotic L. fermentum ME-3 (LfKef) on postprandial OxS, blood TG response and lipoprotein status. 100 clinically healthy subjects were recruited into the study. Blood parameters of postprandial OxS, TG and lipoprotein status were determined by oxidized LDL, baseline diene conjugation in LDL (BDC-LDL), oxidized LDL complex with beta-2 glycoprotein (Beta2-GPI-oxLDL), paraoxonase (PON) activity, LDL-Chol, HDL-Chol and TG. To evaluate general body postprandial OxS-load we measured 8-isoprostanes (8-EPI) in the urine. Consumption of LfKef significantly reduced the postprandial level of oxidized LDL, BDC-LDL, Beta2-GPI-oxLDL, urinary 8-isoprostanes and postprandial TG and caused a significant increase in HDL-Chol and PON activity. This is the first evidence that kefir enriched with an antioxidant probiotic may have a positive effect on both postprandial OxS and TG response as well as on lipoprotein status.     

Changes in the water level of Lake Peipsi and their reflection in a sediment core

A comprehensive study (chronological, lithological and geochemical) of an 8.5 m postglacial sediment sequence from Lake Peipsi was conducted to elucidate the effects of lake-level changes on the sedimentary environment and biogeochemical dynamics in a large lake. Four lithological units were distinguished in the sediment sequence studied: clayey silt, slightly laminated greyish carbonaceous gyttja, brownish-grey gyttja and dark gyttja. These units indicate that large shifts in sedimentation processes occurred in the past. The sediment data show that fluctuations in water depth had a profound impact on the lake environment, recorded as changes in the lithological composition, phosphorus content of sediments and composition of diatom assemblages. The corresponding changes are best reflected in sediments accumulated during phases of regression when the lake area was also smaller. As the water depth and area of the lake increased, wave-induced erosion and resedimentation smoothed or even disturbed the initial information. The increase of phosphorus content in the unconsolidated, high-porosity surface sediments (0.5 m) is most probably connected with active diffusion and matter exchange between the water-sediment pools.     

Engagement of the ATR-Dependent DNA Damage Response at the Human Papillomavirus 18 Replication Centers during the Initial Amplification

We have previously demonstrated that the human papillomavirus (HPV) genome replicates effectively in U2OS cells after transfection using electroporation. The transient extrachromosomal replication, stable maintenance, and late amplification of the viral genome could be studied for high- and low-risk mucosal and cutaneous papillomaviruses. Recent findings indicate that the cellular DNA damage response (DDR) is activated during the HPV life cycle and that the viral replication protein E1 might play a role in this process. We used a U2OS cell-based system to study E1-dependent DDR activation and the involvement of these pathways in viral transient replication. We demonstrated that the E1 protein could cause double-strand DNA breaks in the host genome by directly interacting with DNA. This activity leads to the induction of an ATM-dependent signaling cascade and cell cycle arrest in the S and G₂ phases. However, the transient replication of HPV genomes in U2OS cells induces the ATR-dependent pathway, as shown by the accumulation of γH2AX, ATR-interacting protein (ATRIP), and topoisomerase IIβ-binding protein 1 (TopBP1) in viral replication centers. Viral oncogenes do not play a role in this activation, which is induced only through DNA replication or by replication proteins E1 and E2. The ATR pathway in viral replication centers is likely activated through DNA replication stress and might play an important role in engaging cellular DNA repair/recombination machinery for effective replication of the viral genome upon active amplification.     

Regulation of the frontocortical sodium pump by Na+ in Alzheimer's disease: difference from the age-matched control but similarity to the rat model

The Na+ and K+ dependence of the frontocortical Na,K-ATPase in Alzheimer's disease (AD) was compared with that in human control (Co) and rat AD model. In AD, the relationship between the Na/K ratio and the Na,K-ATPase activity showed noticeable left-shift with three-fold increase in the enzyme affinity for Na+ (K(0.5)=10 and 30 mM in AD and Co, respectively). The Na+ dependence of the enzyme in AD showed two different Hill coefficients (n(H)), 1.1 and 0.3, whereas the Co value of n(H) was higher (1.4). The rat AD model generated by ibotenic acid revealed a Na+ dependence similar to AD. The K+ dependence of the Na,K-ATPase showed no significant difference in AD and Co. Compared with Co, AD produced a shift in the break of the Na,K-ATPase Arrhenius plot, suggesting remarkable alterations in the enzyme lipid environment. Our findings support the hypothesis that dysfunction of the Na,K-ATPase in AD is provoked by altered Na+ dependence of the enzyme. An impairment of the pump functionality might serve as an early mechanism of AD that should be interrupted by selective pharmacological agents.     

Methodological and practical aspects of the presentation and interpretation of microscopic charcoal data from lake sediments

Analysis of microscopic charcoal particles is a useful part of palaeoecological research and is mostly used in conjunction with pollen analysis. However, there is considerable variation in the methodology of charcoal analyses. This paper considers various methods for the analysis of microscopic charcoal and the presentation of charcoal data in the context of a study of the upper sediments of two lakes Estonia. The results are evaluated by comparing both the pollen and charcoal data with documentary evidence of forest fires over the past 60 years. Indications of fires both varying in extent and at different distances from the lakes are evaluated in both the pollen and charcoal diagrams. The results suggest that the total area curve for charcoal particles per unit mass of sediment dry matter (cm² g⁻¹) provides the best indicator of forest fires. Fires in the study area are reflected differently in the charcoal and pollen curves. It is suggested that the charcoal data have the potential to indicate disturbance at a greater distance from the coring site than indicated by the pollen data. Received February 5, 2002 / Accepted August 14, 2002     

Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people

The higher counts or particular groups (Firmicutes/Bacteroidetes) of intestinal microbiota are related to host metabolic reactions, supporting a balance of human ecosystem. We further explored whether intestinal lactobacilli were associated with some principal cellular and metabolic markers of blood in 38 healthy >65-year-old persons. The questionnaire, routine clinical and laboratory data of blood indices as much as the oxidized low-density lipoprotein (ox-LDL) and baseline diene conjugates in low-density lipoprotein (BDC-LDL) of blood sera were explored. The PCR-based intestinal Lactobacillus sp. composition and counts of cultivable lactobacilli (LAB) were tested. The facultative heterofermentative lactobacilli (Lactobacillus casei and Lactobacillus paracasei) were the most frequent (89 and 97%, respectively) species found, while Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri were present in almost half of the elderly persons. The number of species simultaneously colonizing the individuals ranged from 1 to 7 (median 4). In elderly consuming probiotics the LAB counts were significantly higher than in these not consuming (median 7.8, range 4.2–10.8 vs. median 6.3, range 3.3–9.7 log cfu/g; p = 0.005), adjusted (OR = 1.71, CI95 1.04–2.82; p = 0.035) for age and body mass index (BMI). The colonization by L. acidophilus was negatively related (r = ?0.367, p = 0.0275) to L. reuteri, staying significant after adjusting for age, sex and BMI (OR = 0.16, CI95 0.04–0.73; p = 0.018). However, the blood glucose concentration showed a tendency for a negative correlation for colonization with Lactobacillus fermentum (r = ?0.309, p = 0.062) adjusted for BMI (Adj. R² = 0.181; p = 0.013) but not for age and sex. The higher white blood cells (WBC) count was positively related (r = 0.434, p = 0.007) to presence of Lactobacillus reuteri adjusted for age, sex and BMI (Adj. R² = 0.193, p = 0.027). The lower values of ox-LDL were predicted by higher counts of cultivable lactobacilli adjusted by sex, age and BMI (r = ?0.389, p = 0.016; Adj. R² = 0.184 p = 0.029). In conclusion, the pilot study of elderly persons shows that the intestinal lactobacilli are tightly associated with WBC count, blood glucose and content of ox-LDL which all serve as risk markers in pathogenesis of inflammation, metabolic syndrome and cardiovascular disease (CVD).     

A new linear cyclin docking motif that mediates exclusively S‐phase CDK‐specific signaling

Cyclin‐dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation‐dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S‐phase CDK (S‐CDK) Clb5/6‐Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine‐tuning of cell cycle events. Using time‐lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation‐driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL‐containing but not of NLxxxL‐containing substrates. The NLxxxL motif was found to govern S‐cyclin‐specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.     

Acute phase proteins and oxidised low-density lipoprotein in association with ischemic stroke subtype, severity and outcome

Objectives: The goal of our study was to investigate the associations of oxidized LDL (apoB100 aldehyde-modified form) and acute phase proteins (fibrinogen, CRP) with acute ischemic stroke severity and outcome.

Materials and Methods: The study included 61 ischemic stroke patients and 64 controls. Strokes were subtyped according to TOAST criteria, the severity and outcome of stroke (at 1 year) were measured.

Results: The mean triglyceride, fibrinogen, CRP and glucose values were significantly higher among cases. The median oxLDL value for patients with large artery atherosclerosis (LAA) type of stroke was significantly higher than for other subtypes. The oxLDL values did not correlate with age, stroke severity and outcome.

Conclusions: Inflammatory markers (fibrinogen and CRP) predicted the stroke severity and outcome whereas elevation of oxLDL levels did not. Our data refer to possibility that there may exist some links between the LAA subtype of stroke and elevated oxLDL (apoB100 aldehyde-modified form).     

Large-scale Metabolomic Profiling Identifies Novel Biomarkers for Incident Coronary Heart Disease

Analyses of circulating metabolites in large prospective epidemiological studies could lead to improved prediction and better biological understanding of coronary heart disease (CHD). We performed a mass spectrometry-based non-targeted metabolomics study for association with incident CHD events in 1,028 individuals (131 events; 10 y. median follow-up) with validation in 1,670 individuals (282 events; 3.9 y. median follow-up). Four metabolites were replicated and independent of main cardiovascular risk factors [lysophosphatidylcholine 18∶1 (hazard ratio [HR] per standard deviation [SD] increment = 0.77, P-value<0.001), lysophosphatidylcholine 18∶2 (HR = 0.81, P-value<0.001), monoglyceride 18∶2 (MG 18∶2; HR = 1.18, P-value = 0.011) and sphingomyelin 28∶1 (HR = 0.85, P-value = 0.015)]. Together they contributed to moderate improvements in discrimination and re-classification in addition to traditional risk factors (C-statistic: 0.76 vs. 0.75; NRI: 9.2%). MG 18∶2 was associated with CHD independently of triglycerides. Lysophosphatidylcholines were negatively associated with body mass index, C-reactive protein and with less evidence of subclinical cardiovascular disease in additional 970 participants; a reverse pattern was observed for MG 18∶2. MG 18∶2 showed an enrichment (P-value = 0.002) of significant associations with CHD-associated SNPs (P-value = 1.2×10⁻⁷ for association with rs964184 in the ZNF259/APOA5 region) and a weak, but positive causal effect (odds ratio = 1.05 per SD increment in MG 18∶2, P-value = 0.05) on CHD, as suggested by Mendelian randomization analysis. In conclusion, we identified four lipid-related metabolites with evidence for clinical utility, as well as a causal role in CHD development.