Beata Gajewska

Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the growth of Saccharomyces cerevisiae cells deficient in Rsp5 ubiquitin ligase

The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.

Plasma profiling reveals three proteins associated to amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease leading to muscular paralysis and death within 3–5 years from onset. Currently, there are no reliable and sensitive markers able to substantially shorten the diagnosis delay. The objective of the study was to analyze a large number of proteins in plasma from patients with various clinical phenotypes of ALS in search for novel proteins or protein profiles that could serve as potential indicators of disease.

Kinesin Expression in the Central Nervous System of Humans and Transgenic hSOD1G93A Mice with Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis is a fatal motor neuron degenerative disease. Most cases are sporadic (SALS), and approximately 10% are familial (FALS) among which over 20% are linked to the SOD1 mutation. Both SALS and FALS have been associated with retrograde axonal transport defects. Kinesins (KIFs) are motor proteins involved mainly in anterograde transport; however, some also participate in retrograde transport. Objective: The purpose of the study was to investigate and compare the expression of kinesins involved in anterograde (KIF5A, 5C) and retrograde (KIFC3/C2) axonal transport in SALS in humans and FALS in mice with the hSOD1G93A mutation. Methods: The studies were conducted on various parts of the CNS from autopsy specimens of SALS patients, and transgenic mice at presymptomatic and symptomatic stages using real-time quantitative PCR and reverse-transcription PCR. Results: All KIF expression in the motor cortex of individual SALS subjects was higher than in the adjacent sensory cortex, in contrast to the expression in control brains. It was also significantly higher in the frontal cortex of symptomatic but not presymptomatic mice compared to wild-type controls. However, the mean KIF expression in the SALS motor and sensory cortexes was lower than in control cortexes. To a lesser extent the decrease in KIF mean expression also occurred in human but not in mouse ALS spinal cords and in both human and mouse cerebella. Conclusion: Disturbances in kinesin expression in the CNS may dysregulate both anterograde and retrograde axonal transports leading to motor neuron degeneration.

GSTP1 Polymorphisms and their Association with Glutathione Transferase and Peroxidase Activities in Patients with Motor Neuron Disease

Glutathione S-transferase pi (GSTP1) is a crucial enzyme in detoxification of electrophilic compounds and organic peroxides. Together with Se-dependent glutathione peroxidase (Se-GSHPx) it protects cells against oxidative stress which may be a primary factor implicated in motor neuron disease (MND) pathogenesis. We investigated GSTP1 polymorphisms and their relationship with GST and Se-GSTPx activities in a cohort of Polish patients with MND. Results were correlated with clinical phenotypes. The frequency of genetic variants for GSTP1 exon 5 (I105V) and exon 6 (A114V) was studied in 104 patients and 100 healthy controls using real-time polymerase chain reaction. GST transferase activity was determined in serum with 1-chloro-2,4-dinitrobenzene, its peroxidase activity with cumene hydroperoxide, and Se-GSHPx activity with hydrogen peroxide. There were no differences in the prevalence of GSTP1 polymorphism I105V and A114V between MND and controls, however the occurrence of CT variant in codon 114 was associated with a higher risk for MND. GSTP1 polymorphisms were less frequent in classic ALS than in progressive bulbar palsy. In classic ALS C* (heterozygous I /V and A /V) all studied activities were significantly lower than in classic ALS A* (homozygous I /I and A/A). GST peroxidase activity and Se-GSHPx activity were lower in classic ALS C* than in control C*, but in classic ALS A* Se-GSHPx activity was significantly higher than in control A*. It can be concluded that the presence of GSTP1 A114V but not I105V variant increases the risk of MND, and combined GSTP1 polymorphisms in codon 105 and 114 may result in lower protection of MND patients against the toxicity of electrophilic compounds, organic and inorganic hydroperoxides.

Validation of qPCR reference genes in lymphocytes from patients with amyotrophic lateral sclerosis

Quantitative polymerase chain reaction (qPCR) is the most specific and reliable method for determination of mRNA gene expression. Crucial point for its accurate normalization is the choice of appropriate internal control genes (ICGs). In the present work we determined and compare the expression of eight commonly used ICGs in lymphocytes from 26 patients with amyotrophic lateral sclerosis (ALS) and 30 control subjects. Peripheral blood mononuclear cells (PBMCs) before and after immortalization by EBV transfection (lymphoblast cell lines—LCLs) were used for qPCR analysis. LCLs were studied before and after liquid nitrogen cryopreservation and culturing (groups LCL1 and LCL2, respectively). qPCR data of 8 ICGs expression was analyzed by BestKeeper, NormFinder and geNorm methods. All studied genes (18SRNA, ACTB, B2M, GUSB,GAPDH, HPRT1, MT-ATP6 and RPS17) were expressed in PBMCs, whereas only first four in LCLs. LCLs cryopreservation had no effect on ICGs expression. Comprehensive ranking indicated RPS17 with MT-ATP6 as the best ICGs for qPCR in PBMCs of control and ALS subjects, and RPS17 with 18RNA or MT-ATP6 in LCLs from ALS. In PBMCs 18RNA shouldn’t be used as ICG.

Thiram modulates pro-inflammatory mediators in RAW 264.7 murine macrophage cells

Abstract Thiram (TMTD) is a widely used dithiocarbamate pesticide and fungicide and is one of potent contact allergens. In the light of known properties, thiram is also considered to be used as an inhibitor of inflammation. To investigate whether known pro-oxidative properties of thiram might be involved in immunogenic mechanisms, we carried out an in vitro study aimed at analysis of reactive oxygen species (ROS) generation, activation of NF-κB, expression of iNOS and COX-2, production of NO, PGE2 and IL-1β in murine macrophage cells (RAW 264.7). The cells were treated by thiram alone (0.5 µg/mL; 2 μM and 2 µg/mL; 8 μM) or concomitantly with bacterial endotoxin (LPS; 1 μg/mL). LPS was used as an endotoxin that triggers changes characteristic for inflammatory state of the cell. TMTD increased ROS production, level of oxidized glutathione (GSSG) and activated NF-κB. The consequence of NF-κB activation was the increase of IL-1β and NO production characteristic for inflammation. However, we did not observe changes in PGE2 concentration. We observed expression of iNOS, COX-2 proteins and NO and PGE2 production in macrophages treated with thiram concomitantly with LPS lower than those in cells stimulated with LPS alone. Thiram (2 µg/mL) decreased NF-κB activation and production of LPS-induced IL-1β. In conclusion, we demonstrated changes induced by TMTD characteristic for inflammation. Hence, it can be supposed that they may participate in the elicitation phase of allergic contact dermatitis induced by thiram. However, when TMTD acts concomitantly with LPS, it decreases the intensity of inflammation state in RAW 264.7.

ERCC1-deficient cells and mice are hypersensitive to lipid peroxidation

Abstract Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1‐XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO‐induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1−/− mouse embryonic fibroblasts were more sensitive than wild‐type (WT) cells to the LPO products: 4‐hydroxy‐2‐nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1‐XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair‐deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1−/− cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error‐prone translesion DNA synthesis and cellular senescence much more potently in Ercc1−/− cells than in DNA repair‐competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1‐deficient cells and mice are hypersensitive to LPO implicates LPO‐induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats. Graphical abstract Figure. No Caption available. HighlightsERCC1‐deficient mice have increased oxidative stress and LPO.LPO induced DNA and protein damage causes senescence and necrosis in Ercc1−/− cells.HNE induces promutagenic imbalance in BER and TLS in Ercc1−/− mice.Differential antioxidative defense and energy production in Ercc1−/− cells.LPO contributes to premature aging and morbidity.

Alteration of Motor Protein Expression Involved in Bidirectional Transport in Peripheral Blood Mononuclear Cells of Patients with Amyotrophic Lateral Sclerosis

Background: Sporadic amyotrophic lateral sclerosis (SALS) is a fatal motor neuron degenerative disease of unclear pathogenesis. Disturbances of intracellular transport are possible causes of the disease. Objective: We evaluated the expression of motor proteins involved in the anterograde (kinesins KIF1B, KIF5C) and retrograde (KIFC3, dynactin subunits DCTN1 and DCTN3) intracellular transport in peripheral blood mononuclear cells (PBMCs). Materials and Methods: PBMCs were obtained from 74 SALS patients with different clinical phenotypes, 65 blood donors (healthy control I), and 29 cases with other neurological diseases (disease control II) divided into subgroups IIA (atypical parkinsonism) and IIB (ALS-mimicking disorders). mRNA expression was studied by real-time qPCR, and protein level by Western blotting. Results: In SALS, KIF5C and KIFC3 expression was significantly lower and DCTN1 higher than in control I, and dependent of age. KIF1B expression was significantly higher in SALS than in subgroup IIB, whereas DCTN1 and DCTN3 were higher in SALS than in subgroup IIA. All changes in the studied proteins were statistically significant in classic ALS but not in progressive muscular atrophy. Conclusion: In SALS, and especially in classic ALS, the changes in motor protein expression may alter bidirectional intracellular transport in PBMCs. More studies are needed to find out whether the levels of KIF5C and DCTN1 may be useful in ALS diagnosis, and whether KIF1B expression may discriminate ALS from ALS-mimicking disorders.

The potential role of NF-kB activation and synthesis of pro-inflammatory mediators in the pathogenesis of allergic contact dermatitis induced by thiuram

Among the various substances known to cause occupational allergic contact dermatitis, additives to rubber, especially thiurams are regarded as the most important class of contact allergens. It has been suggest that in the sensitization phase of contact hypersensitivity hapten application induces strong innate immune mechanisms, causing the release of danger signals as reactive oxygen species (ROS) generation and activation of NF-eB leading to IL-1β and other cytokine release. To investigate whether pro-oxidative properties of thiuram might be involved in immunogenic mechanisms we analyzed of ROS generation, activation of NF-eB, expression of IL-1â in murine macrophages model. Murine macrophages were treated by non-toxic concentrations of thiuram which induced oxidative stress as was demonstrated by increased production of ROS, especially superoxide anion. Laser scanning confocal microscopy revealed translocation of p65 subunit of NF-eB from cytoplasm to nucleus characteristically for activation NF-eB. The consequence of NF-eB activation was the increase of IL-1â production. These results indicate that thiuram induces changes characteristic for inflammation therefore it can be supposed that they participate in the pathogenesis of allergic contact dermatitis induced by thiuram.

[Intracellular transport proteins: classification, structure and function of kinesins].

Correct cell functioning, division and morphogenesis rely on efficient intracellular transport. Apart from dyneins and myosins, kinesins are the main proteins responsible for intracellular movement. Kinesins are a large, diverse group of motor proteins, which based on phylogenetic similarity were classified into fourteen families. Among these families, due to the location of their motor domains, three groups have been characterized: N-, C- and M-kinesin. As molecular motors, kinesins transport various molecules and vesicles mainly towards the microtubule plus end (from the cell body) participating in anterograde transport, although there are also kinesins involved in retrograde transport (C-kinesins). Kinesins are also involved in spindle formation, chromosome segregation, and spermatogenesis. Because of their great importance for the correct functioning of cells, mutations in kinesin coding genes may lead to such neurodegenerative diseases as dominant hereditary spastic paraplegia or Charcot-Marie-Tooth disease.