Urszula Wojda

Interaction of calcyclin and its cyanogen bromide fragments with annexin II and glyceraldehyde 3-phosphate dehydrogenase.

The structural properties of calcyclin protein are quite well characterized but its function remains obscure. To help elucidate the biological role of calcyclin we have performed the in vitro studies of the Ca(2+)-dependent interaction of Ehrlich ascites tumor cells calcyclin and its cyanogen bromide fragments with two potential calcyclin targets: annexin II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The binding of annexin II, evidenced by the reaction with 125I-calcyclin, was found to be very weak and occurred only for intact calcyclin. On the other hand the interaction between calcyclin and GAPDH was of high affinity and could be assigned to the N-terminal region of calcyclin. Intact calcyclin and its N-terminal fragment bound to GAPDH in the gel overlay and affinity chromatography assay. When examined in the presence of a crosslinking agent the interaction resulted in the formation of 46K covalent adduct between calcyclin monomer and GAPDH subunit. Fluorescence of 5-iodoacetamido-fluorescein-labelled calcyclin was efficiently quenched by GAPDH in the presence of Ca2+. Titration experiments revealed the stoichiometry of one calcyclin monomer binding to each of GAPDH subunits with a binding constant of 10(8) M-1. The results of this work suggest that the binding between calcyclin and GAPDH may have bearing on calcyclin function.

T Lymphocytes and Inflammatory Mediators in the Interplay between Brain and Blood in Alzheimer’s Disease: Potential Pools of New Biomarkers

Alzheimers disease (AD) is a chronic neurodegenerative disorder and the main cause of dementia. The disease is among the leading medical concerns of the modern world, because only symptomatic therapies are available, and no reliable, easily accessible biomarkers exist for AD detection and monitoring. Therefore extensive research is conducted to elucidate the mechanisms of AD pathogenesis, which seems to be heterogeneous and multifactorial. Recently much attention has been given to the neuroinflammation and activation of glial cells in the AD brain. Reports also highlighted the proinflammatory role of T lymphocytes infiltrating the AD brain. However, in AD molecular and cellular alterations involving T cells and immune mediators occur not only in the brain, but also in the blood and the cerebrospinal fluid (CSF). Here we review alterations concerning T lymphocytes and related immune mediators in the AD brain, CSF, and blood and the mechanisms by which peripheral T cells cross the blood brain barrier and the blood-CSF barrier. This knowledge is relevant for better AD therapies and for identification of novel biomarkers for improved AD diagnostics in the blood and the CSF. The data will be reviewed with the special emphasis on possibilities for development of AD biomarkers.

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg(2+)-ATPase activity of myosin in the presence of Ca2+.

Calcyclin from mouse Ehrlich ascites tumor cells and rabbit lung form non-covalent dimers

Crosslinking treatments of fresh cytosol from mouse Ehrlich ascites tumor (EAT) cells revealed the existence of calcyclin dimers which were sensitive to SDS, but not to reducing agents, which suggests the existence of non-covalent dimers. In stored EAT cell cytosol and preparations of purified calcyclin dimers were also formed by S-S bridging (covalent dimers). The S-S dimers did not bind to organomercurial Agarose and could be separated from reduced forms of calcyclin that bound to the resin. Calcyclin eluted from the resin with DTT was a mixture of monomers and non-covalent dimers as shown by crosslinking and subsequent immunoblotting. Calcyclin from rabbit lung, lacking a cysteine residue, could also be crosslinked as a dimer. It is suggested that the ability of calcyclin to form non-covalent dimers is of physiological significance.

Alzheimer's disease lymphocytes: potential for biomarkers?

Alzheimer’s disease: challenges & concepts The neurodegenerative disease described by Alois Alzheimer in 1906 is now recognized as a major aging-related dementia worldwide, affecting more than 35 million individuals. With the demographic shift toward aging societies, this number is expected to double every 20 years, to an estimated 135.5 million worldwide afflicted with the disease by 2050 [1]. The global costs related to diminished social functioning in AD patients are similar to the financial burdens of heart disease and cancer, placing Alzheimer’s disease (AD) among major public health concerns [2]. AD develops for tens of years in the pre

Characterization of calcyclin fragments obtained by CNBr-cleavage

1. Two calcyclin fragments were obtained by CNBr-cleavage. 2. One fragment represented N-terminal end of a molecule (residues 1-56), and another one a C-terminal end (residues 57-89). 3. Properties of intact calcyclin such as binding of calcium, binding to hydrophobic resins and interaction with calcyclin specific antibodies were not retained by these fragments. 4. However, both fragments were able to form dimers and higher forms of aggregates as seen for uncleaved calcyclin. 5. This indicates that both halves of the molecule contain the regions responsible for non-covalent interaction which might participate in dimer formation.

Familial Alzheimer’s Disease Lymphocytes Respond Differently Than Sporadic Cells to Oxidative Stress: Upregulated p53-p21 Signaling Linked with Presenilin 1 Mutants

Familial (FAD) and sporadic (SAD) Alzheimer’s disease do not share all pathomechanisms, but knowledge on their molecular differences is limited. We previously reported that cell cycle control distinguishes lymphocytes from SAD and FAD patients. Significant differences were found in p21 levels of SAD compared to FAD lymphocytes. Since p21 can also regulate apoptosis, the aim of this study was to compare the response of FAD and SAD lymphocytes to oxidative stress like 2-deoxy-D-ribose (2dRib) treatment and to investigate the role of p21 levels in this response. We report that FAD cells bearing seven different PS1 mutations are more resistant to 2dRib-induced cell death than control or SAD cells: FAD cells showed a lower apoptosis rate and a lower depolarization of the mitochondrial membrane. Despite that basal p21 cellular content was lower in FAD than in SAD cells, in response to 2dRib, p21 mRNA and protein levels significantly increased in FAD cells. Moreover, we found a higher cytosolic accumulation of p21 in FAD cells. The transcriptional activation of p21 was shown to be dependent on p53, as it can be blocked by PFT-α, and correlated with the increased phosphorylation of p53 at Serine 15. Our results suggest that in FAD lymphocytes, the p53-mediated increase in p21 transcription, together with a shift in the nucleocytoplasmic localization of p21, confers a survival advantage against 2dRib-induced apoptosis. This compensatory mechanism is absent in SAD cells. Thus, therapeutic and diagnostic designs should take into account possible differential apoptotic responses in SAD versus FAD cells.

Search for Alzheimer's disease biomarkers in blood cells: hypotheses-driven approach

Current Alzheimers disease (AD) diagnostics is based on cognitive testing, and detecting amyloid Aβ and τ pathology by brain imaging and assays of cerebrospinal fluid. However, biomarkers identifying complex pathways contributing to pathology are lacking, especially for early AD. Preferably, such biomarkers should be more cost-effective and present in easily available diagnostic tissues, such as blood. Here, we summarize the recent findings of potential early AD molecular diagnostic biomarkers in blood platelets, lymphocytes and erythrocytes. We review molecular alterations which refer to such main hypotheses of AD pathogenesis as amyloid cascade, oxidative and mitochondrial stress, inflammation and alterations in cell cycle regulatory molecules. The major advantage of such biomarkers is the potential ability to indicate individualized therapies in AD patients.

Oxidant/Antioxidant Imbalance in Alzheimer’s Disease: Therapeutic and Diagnostic Prospects

Alzheimers disease (AD) is the most common cause of dementia and a great socioeconomic burden in the aging society. Compelling evidence demonstrates that molecular change characteristics for AD, such as oxidative stress and amyloid β (Aβ) oligomerization, precede by decades the onset of clinical dementia and that the disease represents a biological and clinical continuum of stages, from asymptomatic to severely impaired. Nevertheless, the sequence of the early molecular alterations and the interplay between them are incompletely understood. This review presents current knowledge about the oxidative stress-induced impairments and compromised oxidative stress defense mechanisms in AD brain and the cross-talk between various pathophysiological insults, with the focus on excessive reactive oxygen species (ROS) generation and Aβ overproduction at the early stages of the disease. Prospects for AD therapies targeting oxidant/antioxidant imbalance are being discussed, as well as for the development of novel oxidative stress-related, blood-based biomarkers for early, noninvasive AD diagnostics.

Overactive BRCA1 Affects Presenilin 1 in Induced Pluripotent Stem Cell-Derived Neurons in Alzheimer's Disease

The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimers disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-β. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-β pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.