bluuebunny/crossref_metadata_2025 huggingface dataset:
Probiotics, prebiotics and antioxidants as functional foods.
Authors: Grajek Włodzimierz , Olejnik Anna , Sip Anna
Published: November 2019

The term "functional foods" comprises some bacterial strains and products of plant and animal origin containing physiologically active compounds beneficial for human health and reducing the risk of chronic diseases. Among the best known functional compounds probiotics, prebiotics and natural antioxidants should be given as examples. These substances can be obtained by biotechnological methods and by extraction from plant or animal tissues.
Biotechnology of temperate fruit trees and grapevines.
Authors: Laimer Margit , Mendonça Duarte , Maghuly Fatemeh , Marzban Gorji , Leopold Stephan , Khan Mahmood , Balla Ildiko , Katinger Hermann
Published: November 2019

Challenges concerning fruit trees and grapevines as long lived woody perennial crops require adapted biotechnological approaches, if solutions are to be found within a reasonable time frame. These challenges are represented by the need for correct identification of genetic resources, with the foreseen use either in conservation or in breeding programmes. Molecular markers provide most accurate information and will be the major solution for questions about plant breeders rights. Providing healthy planting material and rapid detection of newly introduced pathogens by reliable methods involving serological and molecular biological tools will be a future challenge of increases importance, given the fact that plant material travels freely in the entire European Union. But also new breeding goals and transgenic solutions are part of the biotechnological benefits, e.g. resistance against biotic and abiotic stress factors, modified growth habits, modified nutritional properties and altered processing and storage qualities. The successful characterization of transgenic grapevines and stone fruit trees carrying genes of viral origin in different vectors constructed under ecological consideration, will be presented. Beyond technical feasibility, efficiency of resistance, environmental safety and Intellectual Property Rights, also public acceptance needs consideration and has been addressed in a specific project. The molecular determination of internal quality parameters of food can also be addressed by the use of biotechnological tools. Patient independent detection tools for apple allergens have been developed and should allow to compare fruits from different production systems, sites, and genotypes for their content of health threatening compounds.
Yeast as a biosensor for antioxidants: simple growth tests employing a Saccharomyces cerevisiae mutant defective in superoxide dismutase.
Authors: Zyracka Ewa , Zadrag Renata , Kozioł Sabina , Krzepiłko Anna , Bartosz Grzegorz , Biliński Tomasz
Published: November 2019

Mutants of Saccharomyces cerevisiae devoid of Cu,Zn-superoxide dismutase are hypersensitive to a range of oxidants, hyperbaric oxygen and hyperosmotic media, show lysine and methionine auxotrophy when grown under the atmosphere of air and have a shortened replicative life span when compared to the wild-type strain. Ascorbate and other antioxidants can ameliorate these defects, which may be a basis of simple tests sensing the presence of antioxidants. In particular, tests of growth on solid medium (colony formation) in the absence of methionine and/or lysine, or in the presence of 0.8 M NaCl can be useful for detection and semiquantitative estimation of compounds of antioxidant properties. Hypoxic atmosphere was found to increase the sensitivity of detection of antioxidants. The test of abolishment of lysine auxotrophy showed a concentration dependence of the antioxidant effects of cysteine and N-acetylcysteine which, however, lost their protective action at high concentration, in contrast to glutathione which was effective also at higher concentrations.
Molecular simulation of adsorption from dilute solutions.
Authors: Billes Werner , Tscheliessnig Rupert , Fischer Johann
Published: November 2019

Adsorption of biomolecules on surfaces is a perennial and general challenge relevant to many fields in biotechnology. A change of the Helmholtz free energy DeltaA takes place when a molecule becomes adsorbed out of a bulk solution. The purpose of our investigations is to explore routes for the calculation of DeltaA by molecular simulations. DeltaA can be obtained both by integration over the mean force on a molecule and via the local density. It turns out that the route via the potential of mean force prevails over the latter due to better consistency. In this work we present results for systems of 1-centre and 2-centre Lennard-Jones mixtures at a 9/3 Lennard-Jones wall.
Molecular basis of cellulose biosynthesis disappearance in submerged culture of Acetobacter xylinum.
Authors: Krystynowicz Alina , Koziołkiewicz Maria , Wiktorowska-Jezierska Agnieszka , Bielecki Stanisław , Klemenska Emilia , Masny Aleksander , Płucienniczak Andrzej
Published: November 2019

Acetobacter xylinum strains are known as very efficient producers of bacterial cellulose which, due to its unique properties, has great application potential. One of the most important problems faced during cellulose synthesis by these bacteria is generation of cellulose non-producing cells, which can appear under submerged culture conditions. The reasons of this remain unknown. These studies have been undertaken to compare at the molecular level wild-type, cellulose producing (Cel(+)) A. xylinum strains with Cel(-) forms of cellulose-negative phenotype. Comparison of protein profiles of both forms of A. xylinum by 2D electrophoresis allowed for the isolation of proteins which were produced exclusively by either Cel+ or Cel- cells. Sequences of peptides derived from these proteins were aligned with those of proteins deposited in databases. This analysis revealed that Cel(-) cells lacked two enzymes: phosphoglucomutase and glucose-1-phosphate uridylyltransferase, which generates UDP-glucose being the substrate for cellulose synthase. DNA was analyzed by ligation-mediated PCR carried out at low denaturation temperature (PCR-MP). Two DNA fragments of different thermal stability (218 and 217 bp) were obtained from the DNA of Cel(+) and Cel(-) forms, respectively. The only difference between these Cel(-) and Cel(+) DNA fragments is deletion of one T residue. Alignment of those two sequences with those deposited in the GenBank database revealed that similar fragments are present in the genomes of some bacterial cellulose producers and are located downstream from open reading frames (ORF) encoding phosphoglucomutase. The meaning of this observation is discussed.
Virus-like particles of potato leafroll virus as potential carrier system for nucleic acids.
Authors: Sułuja Elzbieta , Strokowskaja Ludmiła , Zagórski-Ostoja Włodzimierz , Pałucha Andrzej
Published: November 2019

Potato leafroll virus is a member of the polerovirus genus. The isometric virion is formed by a coat protein encapsidating single-stranded, positive-sense, mono-partite genomic RNA with covalently attached viral protein at the 5' end. The coat protein of the virus exists in two forms: i) a 23 kDa protein, the product of the coat protein gene, and ii) a 78 kDa protein, the product of the coat protein gene and an additional open reading frame expressed by read-through of the coat protein gene stop codon. The aim of this work was the expression of potato leafroll virus coat protein-based proteins that would be able to assemble into virus-like particles in insect cells. These modified particles were tested for their ability to encapsidate nucleic acids. Two types of N-terminally His-tagged coat protein constructs were used for the expression in insect cells: one, encoding a 23 kDa protein with the C-terminal amino-acid sequence corresponding to the wild type coat protein and the second with additional clathrin binding domain at the C-terminus. The expression of these two proteins by a recombinant baculovirus was characterized by Western immunoblotting with antibodies directed against potato leafroll virus. The protection or putative encapsidation of nucleic acids by these two coat protein derivatives was shown by DNase I and RNase A protection assays.
Improved downstream process for the production of plasmid DNA for gene therapy.
Authors: Urthaler Jochen , Buchinger Wolfgang , Necina Roman
Published: November 2019

Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.
Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1.
Authors: Schmid Gerald , Wojciechowski Jacek , Wesierska-Gadek Józefa
Published: November 2019

We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.
High pressure processing for food safety.
Authors: Fonberg-Broczek Monika , Windyga B , Szczawiński J , Szczawińska M , Pietrzak D , Prestamo G
Published: November 2019

Food preservation using high pressure is a promising technique in food industry as it offers numerous opportunities for developing new foods with extended shelf-life, high nutritional value and excellent organoleptic characteristics. High pressure is an alternative to thermal processing. The resistance of microorganisms to pressure varies considerably depending on the pressure range applied, temperature and treatment duration, and type of microorganism. Generally, Gram-positive bacteria are more resistant to pressure than Gram-negative bacteria, moulds and yeasts; the most resistant are bacterial spores. The nature of the food is also important, as it may contain substances which protect the microorganism from high pressure. This article presents results of our studies involving the effect of high pressure on survival of some pathogenic bacteria -- Listeria monocytogenes, Aeromonas hydrophila and Enterococcus hirae -- in artificially contaminated cooked ham, ripening hard cheese and fruit juices. The results indicate that in samples of investigated foods the number of these microorganisms decreased proportionally to the pressure used and the duration of treatment, and the effect of these two factors was statistically significant (level of probability, P
Nutritional properties of tubers of conventionally bred and transgenic lines of potato resistant to necrotic strain of Potato virus Y (PVYN).
Authors: Juśkiewicz Jerzy , Zduńczyk Zenon , Fornal Józef
Published: November 2019

The potential effect of genetic modification on nutritional properties of potatoes transformed to improve resistance to a necrotic strain of Potato virus Y was determined in a rat experiment. Autoclaved tubers from four transgenic lines were included to a diet in the amount of 40% and compared with the conventional cv. Irga. The experiment lasted 3 weeks and special attention was paid to nutritional properties of diets, caecal metabolism and serum indices. Genetic modification of potato had no negative effect on the chemical composition and nutritional properties of tubers, ecosystem of the caecum, activity of serum enzymes and non-specific defence mechanism of the rats. Obtained results indicate that transgenic potato with improved resistance to PVY(N): line R1F (truncated gene coding for PVY(N) polymerase in sense orientation), R2P (truncated gene coding for PVY(N) polymerase in antisense orientation), and NTR1.16 (non-translated regions of PVY(N) genome in sense orientation) are substantial and nutritional equivalence to the non-transgenic cultivar. Tubers of transgenic line NTR2.27 (non-translated regions of PVY(N) genome in antisense orientation) increased the bulk of caecal digesta and the production of SCFA as compared to tubers of the conventional cultivar and the other transgenic clones. Taking into account some deviations, it seems reasonable to undertake a long-term feeding study to confirm the nutritional properties of tubers of transgenic lines.
Retroviral targeting of proliferating endothelial cells.
Authors: Gornikiewicz Alexander , Zommer Anna , Jakesz Raimund , Gnant Michael , Brostjan Christine
Published: November 2019

Tumor growth requires the formation of new blood vessels by endothelial cells. Thus, surface molecules -- such as angiogenin receptors -- that are selectively expressed on growing endothelium represent an attractive target for directed delivery of compounds to tumor tissue. We attempted to obtain genetically engineered retroviral vectors targeted to the endothelium by inserting the human angiogenin sequence into Moloney murine leukemia virus envelope glycoprotein. Abundant expression of the chimeric protein could be verified. However, while being selective for proliferating human endothelial cells, the recombinant retroviral particles displayed low transduction efficiencies and thus have to be further improved.
Targeting drug-efflux pumps -- a pharmacoinformatic approach.
Authors: Pleban Karin , Kaiser Dominik , Kopp Stefan , Peer Michael , Chiba Peter , Ecker Gerhard F
Published: November 2019

In line with our studies on propafenone-type inhibitors of P-glycoprotein (P-gp), we applied several methods to approach virtual screening tools for identification of new P-gp inhibitors on one hand and the molecular basis of ligand-protein interaction on the other hand. For virtual screening, a combination of autocorrelation vectors and selforganising artificial neural networks proved extremely valuable in identifying P-gp inhibitors with structurally new scaffolds. For a closer view on the binding region for propafenone-type ligands we applied a combination of pharmacophore-driven photoaffinity labeling and protein homology modeling. On LmrA, a bacterial homologue of P-gp, we were able to identify distinct regions on transmembrane helices 3, 5 and 6 which show significant changes in the labeling pattern during different steps of the catalytic cycle.
Heme oxygenase-1 expression in disease states.
Authors: Deshane Jessy , Wright Marcienne , Agarwal Anupam
Published: December 2019

Heme oxygenase-1 (HO-1) is an enzyme which catalyzes the rate-limiting step in heme degradation resulting in the formation of iron, carbon monoxide and biliverdin, which is subsequently converted to bilirubin by biliverdin reductase. The biological effects exerted by the products of this enzymatic reaction have gained much attention. The anti-oxidant, anti-inflammatory and cytoprotective functions associated with HO-1 are attributable to one or more of its degradation products. Induction of HO-1 occurs as an adaptive and beneficial response to several injurious stimuli including heme and this inducible nature of HO-1 signifies its importance in several pathophysiological disease states. The beneficial role of HO-1 has been implicated in several clinically relevant disease states involving multiple organ systems as well as significant biological processes such as ischemia-reperfusion injury, inflammation/immune dysfunction and transplantation. HO-1 has thus emerged as a key target molecule with therapeutic implications.
Targeting site-specific chromosome integration.
Authors: Nuno-Gonzalez Patricia , Chao Hsu , Oka Kazuhiro
Published: December 2019

The concept of gene therapy was introduced with great promise and high expectations. However, what appeared simple in theory has not translated into practice. Despite some success in clinical trials, the research community is still facing an old problem: namely, the need for a vector that can deliver a gene to target cells without adverse events while maintaining a long-term therapeutic effect. Some of these challenges are being addressed by the development of hybrid vectors which meld two different viral systems to incorporate efficient gene delivery and large cloning capacity with site-specific integration. The two known systems that integrate genes into specific sites in mammalian genomes are the adeno-associated virus and phage integrases. Recent experiments with hybrid vectors incorporating both of these systems are encouraging. However, extensive research should be directed towards the safety and efficacy of this approach before it will be available for gene therapy.
Adeno-associated virus vector-mediated gene delivery to the vasculature and kidney.
Authors: Kapturczak Matthias H , Chen Sifeng , Agarwal Anupam
Published: December 2019

Relatively successful elsewhere, gene delivery aimed at the vasculature and kidney has made very little progress. In the kidney, the hurdles are related to the unique structure-function relationships of this organ and in the blood vessels to a variety of, mostly endothelial, factors making the delivery of transgenes very difficult. Among gene-therapeutic approaches, most viral gene delivery systems utilized to date have shown significant practical and safety-related limitations due to the level and duration of recombinant transgene expression as well as their induction of a significant host immune response to vector proteins. Recombinant adeno-associated virus (rAAV) vectors appear to offer a vehicle for safe, long-term transgene expression. rAAV-based vectors are characterized by a relative non-immunogenicity and the absence of viral coding sequences. Furthermore, they allow for establishment of long-term latency without deleterious effects on the host cell. This brief review addresses problems related to transgene-delivery to kidney and vasculature with particular attention given to rAAV vectors. The potential for gene therapy as a strategy for selected renal and vascular diseases is also discussed.
Design of vascular endothelium-specific drug-targeting strategies for the treatment of cancer.
Authors: Molema Grietje
Published: December 2019

Tumor endothelial cells are actively involved in the neovascularization processes that accompany tumor growth. Their easy accessibility for systemically applied therapeutics makes them interesting targets for therapeutic intervention. Especially for drug targeting-based therapeutics that often consist of macromolecular moieties, the tumor endothelium is considered a much better target than the tumor cells located behind the vascular wall barrier. In this review, the general principles underlying the development and choices in the development of vascular drug-targeting strategies are discussed. An overview of target epitopes identified in the past two decades is followed by a summary of those strategies that directly or indirectly induced tumor blood flow blockade in vivo. The demonstrated therapeutic success in pre-clinical animal models in debulking large tumor masses and inhibiting tumor outgrowth warrant further development of these therapeutic approaches. Yet, more effort should be put in studies in which the efficacy of different effector activities aimed at the same target, of one effector activity aimed at different targets, and of multiple target strategies are be compared. Combining these data with proper inventories on the molecular basis of tumor endothelial heterogeneity in general will make possible the development of tumor vascular drug-targeting strategies towards clinical application.
Inhibition and regression of atherosclerotic lesions.
Authors: Oka Kazuhiro , Chan Lawrence
Published: December 2019

Atherosclerosis, once believed to be a result of a slow, irreversible process resulting from lipid accumulation in arterial walls, is now recognized as a dynamic process with reversibility. Liver-directed gene therapy for dyslipidemia aims to treat patients who are not responsive to currently available primary and secondary prevention. Moreover, gene therapy strategies have also proved valuable in studying the dynamics of atherosclerotic lesion formation, progression, and remodeling in experimental animals. Recent results on the long-term effect of gene therapy suggest that hepatic expression of therapeutic genes suppresses inflammation and has profound effects on the nature of the atherogenic process.
Prospects for p53-based cancer therapy.
Authors: Stokłosa Tomasz , Gołab Jakub
Published: December 2019

The p53 tumor suppressor plays the role of a cellular hub which gathers stress signals such as damage to DNA or hypoxia and translates them into a complex response. p53 exerts its action mainly as a potent transcription factor. The two major outcomes of p53 activity are highlighted: cell cycle arrest and apoptosis. During malignant transformation p53 or p53-pathway related molecules are disabled extremely often. Mutations in p53 gene are present in every second human tumor. A mutant form of p53 may not only negate the wild type p53 function but may play additional role in tumor progression. Therefore p53 represents a relatively unique and specific target for anticancer drug design. Current approaches include several different molecules able to restore p53 wild-type conformation and activity. Such small molecule drugs hold great promise in treating human tumors with dysfunction of p53 pathway in the near future.
TGF beta signalling and its role in tumour pathogenesis.
Authors: Kaminska Bozena , Wesolowska Aleksandra , Danilkiewicz Malgorzata
Published: December 2019

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation and survival/or apoptosis of many cells. Knock-out experiments in mice for the three isoforms of TGF-beta have demonstrated their importance in regulating inflammation and tissue repair. TGF-beta is implicated in the pathogenesis of human diseases, including tissue fibrosis and carcinogenesis. TGF-beta receptors act through multiple intracellular pathways. Upon binding of TGF-beta with its receptor, receptor-regulated Smad2/3 proteins become phosphorylated and associate with Smad4. Such complex translocates to the nucleus, binds to DNA and regulates transcription of specific genes. Negative regulation of TGF-beta/Smad signalling may occur through the inhibitory Smad6/7. Furthermore, TGF-beta-activated kinase-1 (TAK1) is a component of TGF-beta signalling and activates stress-activated kinases: p38 through MKK6 or MKK3 and c-Jun N-terminal kinases (JNKs) via MKK4. In the brain TGF-beta, normally expressed at the very low level, increases dramatically after injury. Increased mRNA levels of the three TGF-beta isoforms correlate with the degree of malignancy of human gliomas. TGF-betas are secreted as latent precursors requiring activation into the mature form. TGF-beta may contribute to tumour pathogenesis by direct support of tumour growth and influence on local microenvironment, resulting in immunosuppression, induction of angiogenesis, and modification of the extracellular matrix. TGF-beta1,2 may stimulate production of vascular endothelial growth factor (VEGF) as well as plasminogen activator inhibitor (PAI-I), that are involved in vascular remodelling occurring during angiogenesis. Blocking of TGF-beta action inhibits tumour viability, migration, metastases in mammary cancer, melanoma and prostate cancer model. Reduction of TGF-beta production and activity may be a promising target of therapeutic strategies to control tumour growth.
Direct tumor damage mechanisms of photodynamic therapy.
Authors: Nowis Dominika , Makowski Marcin , Stokłosa Tomasz , Legat Magdalena , Issat Tadeusz , Gołab Jakub
Published: December 2019

Photodynamic therapy (PDT) is a clinically approved and rapidly developing cancer treatment regimen. It is a minimally invasive two-stage procedure that requires administration of a photosensitizing agent followed by illumination of the tumor with visible light usually generated by laser sources. A third component of PDT is molecular oxygen which is required for the most effective antitumor effects. In the presence of the latter, light of an appropriate wavelength excites the photosensitizer thereby producing cytotoxic intermediates that damage cellular structures. PDT has been approved in many countries for the treatment of lung, esophageal, bladder, skin and head and neck cancers. The antitumor effects of this treatment result from the combination of direct tumor cell photodamage, destruction of tumor vasculature and activation of an immune response. The mechanisms of the direct photodamage of tumor cells, the signaling pathways that lead to apoptosis or survival of sublethaly damaged cells, and potential novel strategies of improving the antitumor efficacy of PDT are discussed.
Stem cell biology: a never ending quest for understanding.
Authors: Majka Marcin , Kucia Magdalena , Ratajczak Mariusz Z
Published: December 2019

Stem cells (SC) research is an important part of biotechnology that could lead to the development of new therapeutic strategies. A lot of effort has been put to understand biology of the stem cells and to find genes and subsequently proteins that are responsible for their proliferation, self-renewal and differentiation. Different cytokines and growth factors has been used to expand stem cells, but no combination of these factors was identified that could effectively expand the most primitive stem cells. Recently, however, genes and receptors responsible for SC proliferation and differentiation have been described. Ligands for these receptors or these genes themselves are being already used for ex vivo expansion of stem cells and the first data are very promising. New markers, such as CXCR4 and CD133, have been discovered and shown to be present on surface of hematopoietic stem cells. The same markers were recently also found to be expressed on neuronal-, hepatic- or skeletal muscle-stem cells. By employing these markers several laboratories are trying to isolate stem cells for potential clinical use. New characteristics of stem cells such as transdifferentiation and cell fusion have been described. Our team has identified a population of tissue committed stem cells (TCSC). These cells are present in a bone marrow and in other tissues and they can differentiate into several cell types including cardiac, neural and liver cells.
Genetic models to study adult neurogenesis.
Authors: Filipkowski Robert K , Kiryk Anna , Kowalczyk Anna , Kaczmarek Leszek
Published: December 2019

In the central nervous system (CNS) generation of new neurons continues throughout adulthood, when it is limited to the olfactory bulb and hippocampus. The knowledge regarding the function of newly-generated neurons remains limited and is vigorously investigated using diverse approaches. Among these are genetically modified mice, most of them of knock-out type (KO). Results from 23 diverse KO mouse models demonstrate the importance of particular proteins (growth factors, nitric oxide synthases, receptors, cyclins/cyclin-associated proteins, transcription factors, etc.) in adult neurogenesis (ANGE) as well as separate it from developmental neurogenesis. These results bring us closer to revealing the function of newly generated neurons in adult brains.
Structure, function, and regulation of myosin 1C.
Authors: Barylko Barbara , Jung Gwanghyun , Albanesi Joseph P
Published: December 2019

Myosin 1C, the first mammalian single-headed myosin to be purified, cloned, and sequenced, has been implicated in the translocation of plasma membrane channels and transporters. Like other forms of myosin I (of which eight exist in humans) myosin 1C consists of motor, neck, and tail domains. The neck domain binds calmodulins more tightly in the absence than in the presence of Ca(2+). Release of calmodulins exposes binding sites for anionic lipids, particularly phosphoinositides. The tail domain, which has an isoelectic point of 10.5, interacts with anionic lipid headgroups. When both neck and tail lipid binding sites are engaged, the myosin associates essentially irreversibly with membranes. Despite this tight membrane binding, it is widely believed that myosin 1C docking proteins are necessary for targeting the enzyme to specific subcellular location. The search for these putative myosin 1C receptors is an active area of research.
Endoplasmic reticulum quality control and apoptosis.
Authors: Groenendyk Jody , Michalak Marek
Published: December 2019

The ER is one of the most important folding compartments within the cell, as well as an intracellular Ca(2+) storage organelle and it contains a number of Ca(2+) regulated molecular chaperones responsible for the proper folding of glycosylated as well as non-glycosylated proteins. The luminal environment of the ER contains Ca(2+) which is involved in regulating chaperones such as calnexin and calreticulin, as well as apoptotic proteins caspase-12 and Bap31, which may play an important role in determining cellular sensitivity to ER stress and apoptosis. The ER quality control system consists of several molecular chaperones, including calnexin, that assist in properly folding proteins and transporting them through the ER as well as sensing misfolded proteins, attempting to refold them and if this is not possible, targeting them for degradation. Accumulation of misfolded protein in the ER leads to activation of genes responsible for the expression of ER chaperones. The UPR mechanism involves transcriptional activation of chaperones by the membrane-localized transcription factor ATF6, in conjunction with the ER membrane kinase IRE1, as well as translational repression of protein synthesis by another ER membrane kinase PERK. When accumulation of misfolded protein becomes toxic, apoptosis is triggered, potentially with IRE1 involved in signaling via caspase-12. Both the extrinsic and intrinsic apoptotic pathways appear to culminate in the activation of caspases and this results in the recruitment of mitochondria in an essential amplifying manner. Bap31 may direct pro-apoptotic crosstalk between the ER and the mitochondria via Ca(2+) in conjunction with caspase-12 and calnexin. Accordingly, ER stress and the resultant Ca(2+) release must be very carefully regulated because of their effects in virtually all areas of cell function.
Store-operated calcium entry in physiology and pathology of mammalian cells.
Authors: Targos Berenika , Barańska Jolanta , Pomorski Paweł
Published: December 2019

One of the numerous calcium-involving processes in mammalian cells is store-operated calcium entry (SOCE) -- the process in which depletion of calcium stores in the endoplasmic reticulum (ER) induces calcium influx from the extracellular space. Previously supposed to function only in non-excitable cells, SOCE is now known to play a role also in such excitable cells as neurons, muscles and neuroendocrine cells and is found in many different cell types. SOCE participates not only in processes dependent on ER calcium level but also specifically regulates some important processes such as cAMP production, T lymphocyte activation or induction of long-term potentiation. Impairment of SOCE can be an element of numerous disorders such as acute pancreatitis, primary immunodeficiency and, since it can take part in apoptosis or cell cycle regulation, SOCE may also be partially responsible for such serious disorders as Alzheimer disease and many types of cancer. Even disturbances in the 'servant' role of maintaining ER calcium level may cause serious effects because they can lead to ER homeostasis disturbance, influencing gene expression, protein synthesis and processing, and the cell cycle.
Molecular mechanisms initiating amyloid beta-fibril formation in Alzheimer's disease.
Authors: Kirkitadze Marina D , Kowalska Anna
Published: December 2019

The deposition of aggregated amyloid beta-protein (Abeta) in the human brain is a major lesion in Alzheimer' disease (AD). The process of Abeta fibril formation is associated with a cascade of neuropathogenic events that induces brain neurodegeneration leading to the cognitive and behavioral decline characteristic of AD. Although a detailed knowledge of Abeta assembly is crucial for the development of new therapeutic approaches, our understanding of the molecular mechanisms underlying the initiation of Abeta fibril formation remains very incomplete. The genetic defects responsible for familial AD influence fibrillogenesis. In a majority of familial cases determined by amyloid precursor protein (APP) and presenilin (PS) mutations, a significant overproduction of Abeta and an increase in the Abeta42/Abeta40 ratio are observed. Recently, it was shown that the two main alloforms of Abeta have distinct biological activity and behaviour at the earliest stage of assembly. In vitro studies demonstrated that Abeta42 monomers, but not Abeta40, form initial and minimal structures (pentamer/hexamer units called paranuclei) that can oligomerize to larger forms. It is now apparent that Abeta oligomers and protofibrils are more neurotoxic than mature Abeta fibrils or amyloid plaques. The neurotoxicity of the prefibrillar aggregates appears to result from their ability to impair fundamental cellular processes by interacting with the cellular membrane, causing oxidative stress and increasing free Ca(2+) that eventually lead to apoptotic cell death.
Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants.
Authors: Juszczyk Paulina , Kołodziejczyk Aleksandra S , Grzonka Zbigniew
Published: December 2019

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.
Splenic eumelanin differs from hair eumelanin in C57BL/6 mice.
Authors: Plonka Przemyslaw M , Michalczyk Dominika , Popik Malgorzata , Handjiski Bori , Slominski Andrzej , Paus Ralf
Published: December 2019

The presence of melanin in spleens of black C57BL/6 mice has been known for long. Although its origin and biological functions are still obscure, the relation of splenic melanin to the hair follicle and skin pigmentation was suggested. Here, we demonstrated using for the first time electron paramagnetic resonance spectroscopy that black-spotted C57BL/6 spleens contain eumelanin. Its presence here is a "yes or no" phenomenon, as even in the groups which revealed the highest percentage of spots single organs completely devoid of the pigment were found. Percentage of the spotted spleens decreased, however, with the progress of telogen after spontaneously-induced hair growth. The paramagnetic properties of the spleen eumelanin differed from the hair shaft or anagen VI skin melanin. The splenic melanin revealed narrower signal, and its microwave power saturability betrayed more heterogenous population of paramagnetic centres than in the skin or hair shaft pigment. Interestingly, the pigment of dry hair shafts and of the wet tissue of depilated anagen VI skin revealed almost identical properties. The properties of splenic melanin better resembled the synthetic dopa melanin (water suspension, and to a lesser degree -- powder sample) than the skin/hair melanin. All these findings may indicate a limited degradation of splenic melanin as compared to the skin/hair pigment. The splenic eumelanin may at least in part originate from the skin melanin phagocyted in catagen by the Langerhans cells or macrophages and transported to the organ.
The involvement of oxidative stress in determining the severity and progress of pathological processes in dystrophin-deficient muscles.
Authors: Niebrój-Dobosz Irena , Hausmanowa-Petrusewicz Irena
Published: December 2019

In both forms of muscular dystrophy, the severe Duchenne's muscular dystrophy (DMD) with lifespan shortened to about 20 years and the milder Becker dystrophy (BDM) with normal lifespan, the gene defect is located at chromosome locus Xp21. The location is the same in the experimental model of DMD in the mdx mice. As the result of the gene defect a protein called dystrophin is either not synthesized, or is produced in traces. Although the structure of this protein is rather well established there are still many controversies about the dystrophin function. The most accepted suggestion supposes that it stabilizes sarcolemma in the course of the contraction-relaxation cycle. Solving the problem of dystrophin function is a prerequisite for introduction of an effective therapy. Among the different factors which might be responsible for the appearance and progress of dystrophic changes in muscles there is an excessive action of oxidative stress. In this review data indicating the influence of oxidative stress on the severity of the pathologic processes in dystrophy are discussed. Several pieces of data indicating the action of oxidative damage to different macromolecules in DMD/BDM are presented. Special attention is devoted to the degree of oxidative damage to muscle proteins, the activity of neuronal nitric oxide synthase (nNOS) and their involvement in defining the severity of the dystrophic processes. It is indicated that the severity of the morbid process is related to the degree of oxidative damage to muscle proteins and the decrease of the nNOS activity in muscles. Estimation of the degree of the destructive action of oxidative stress in muscular dystrophy may be a useful marker facilitating introduction of an effective antioxidant therapy and regulation of nNOS activity.
Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice.
Authors: Jakubiec-Puka Anna , Biral Donatella , Krawczyk Kazimierz , Betto Romeo
Published: December 2019

alpha-Sarcoglycan is a 50 kDa single-pass transmembrane glycoprotein exclusively expressed in striated muscle that, together with beta-, gamma-, and delta-sarcoglycan, forms a sub-complex at the muscle fibre cell membrane. The sarcoglycans are components of the dystrophin-associated glycoprotein (DAG) complex which forms a mechanical link between the intracellular cytoskeleton and extracellular matrix. The DAG complex function is to protect the muscle membrane from the stress of contractile activity and as a structure for the docking of signalling proteins. Genetic defects of DAG components cause muscular dystrophies. A lack or defects of alpha-sarcoglycan causes the severe type 2D limb girdle muscular dystrophy. alpha-Sarcoglycan-null (Sgca-null) mice develop progressive muscular dystrophy similar to the human disorder. This animal model was used in the present work for an ultrastructural study of diaphragm muscle. Diaphragm from Sgca-null mouse presents a clear dystrophic phenotype, with necrosis, regeneration, fibre hypertrophy and splitting, excess of collagen and fatty infiltration. Some abnormalities were also observed, such as centrally located nuclei of abnormal shape, fibres containing inclusion bodies within the contractile structure, and fibres with electron-dense material dispersed over almost the entire cell. Additionally, unusual interstitial cells of uncertain identity were detected within muscle fibres. The abnormal ultrastructure of the diaphragm from Sgca-null mice is discussed.
Beta-actin in human colon adenocarcinoma cell lines with different metastatic potential.
Authors: Nowak Dorota , Skwarek-Maruszewska Aneta , Zemanek-Zboch Magdalena , Malicka-Błaszkiewicz Maria
Published: December 2019

Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between beta-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105000 x g), isolated from the tumor cells was used as a source for actin quantification. The higher level of beta-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of beta-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. beta-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-beta actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) beta-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and beta-actin expression.
Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form.
Authors: Sobczak Adam , Blazejczyk Magdalena , Piszczek Grzegorz , Zhao Gang , Kuznicki Jacek , Wojda Urszula
Published: December 2019

The EF-hand Ca(2+)-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca(2+) signals. As oligomerization may represent one of Ca(2+)-signal transduction mechanisms, we characterised recombinant calmyrin forms using non-reducing SDS/PAGE, analytical ultracentrifugation and gel filtration. We also aimed at identification of biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca(2+)-bound calmyrin oligomerizes forming stable intermolecular disulfide bridges. Ultracentrifugation indicated that at a 220 microM initial protein concentration apo-calmyrin existed in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer (trimeric or tetrameric species were not detected). The dimerization constant was calculated as Ka = 1.78 x 103 M(-1) at 6 degrees C. Gel filtration of apo- and Ca(2+)-bound calmyrin at a 100 microM protein concentration confirmed an equilibrium of a monomer and a covalent dimer state. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca(2+). However, when calmyrin forms were analyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brain. Moreover, in contrast to recombinant calmyrin, crosslinking did not preserve any dimeric species of calmyrin regardless of Ca(2+) concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.
A 2D-IR study of heat- and [(13)C]urea-induced denaturation of sarcoplasmic reticulum Ca(2+)-ATPase.
Authors: Iloro Ibón , Goñi Félix M , Arrondo José L R
Published: December 2019

Two-dimensional infrared correlation spectroscopy (2D-IR) was applied to the study of urea- and heat-induced unfolding denaturation of sarcoplasmic reticulum Ca(2+)-ATPase (SR ATPase). Urea at 2-3 M causes reversible loss of SR ATPase activity, while higher concentrations induce irreversible denaturation. Heat-induced denaturation is a non-two-state process, with an "intermediate state" (at t approximately 45 degrees C) characterized by the presence of protein monomers, instead of the native oligomers. 2D-IR reveals that urea denaturation causes loss of the structural transition to the "intermediate state". Whenever the urea effect can be reversed, the transition to the "intermediate state" is re-established.