Publications by authors named "Magda Dubińska-Magiera"

28 Publications

  • Page 1 of 1

Characterization of Hspb8 in Zebrafish.

Cells 2020 06 26;9(6). Epub 2020 Jun 26.

Department of Animal Developmental Biology, Faculty of Biological Sciences, University of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw, Poland.

Hspb8 is a member of the small heat shock protein (sHSP) family. Its expression is known to be upregulated under heat shock. This protein interacts with different partners and can, therefore, be involved in various processes relevant to tissue integrity and functioning. In humans, mutations in the gene encoding Hspb8 can lead to the development of various diseases such as myopathies and neuropathies. In our study, we aimed to perform an in-depth characterization of zebrafish Hspb8 during zebrafish development. We applied techniques such as RT-qPCR, Western blot, immunofluorescence, co-immunoprecipitation, LC-MS, and morpholino-mediated knockdown. We broadened the knowledge regarding zebrafish expression during development under normal and heat shock conditions as well as its tissue- and subcellular-specific localization. A co-IP analysis allowed us to conclude that zebrafish Hspb8 can interact with proteins such as Bag3 and Hsc70, which are crucial for formation of an autophagy-inducing complex. We also demonstrated that morpholino-mediated knockdown has an impact on zebrafish embryos' morphology, muscle ultrastructure, and motility behavior. Our research provides a valuable resource for the potential use of the zebrafish as a model for studying pathological conditions associated with disorders.
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http://dx.doi.org/10.3390/cells9061562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348923PMC
June 2020

Correction to: Xenopus LAP2β protein knockdown affects location of Lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

Protoplasma 2020 01;257(1):331-332

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, Joliot- Curie 14a, 50-383, Wrocław, Poland.

The original publication of this paper contains a mistake.
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http://dx.doi.org/10.1007/s00709-019-01474-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050555PMC
January 2020

Everybody wants to move-Evolutionary implications of trunk muscle differentiation in vertebrate species.

Semin Cell Dev Biol 2020 08 20;104:3-13. Epub 2019 Nov 20.

Department of Animal Developmental Biology, Institute of Experimental Biology, University of Wroclaw, Sienkiewicza 21, 50-335 Wrocław, Poland.

In our review we have completed current knowledge on myotomal myogenesis in model and non-model vertebrate species (fishes, amphibians, reptiles, birds and mammals) at morphological and molecular levels. Data obtained from these studies reveal distinct similarities and differences between amniote and anamniote species. Based on the available data, we decided to present evolutionary implications in vertebrate trunk muscle development. Despite the fact that in all vertebrates muscle fibres are multinucleated, the pathways leading to them vary between vertebrate taxa. In fishes during early myogenesis myoblasts differentiate into multinucleated lamellae or multinucleate myotubes. In amphibians, myoblasts fuse to form multinucleated myotubes or, bypassing fusion, directly differentiate into mononucleated myotubes. Furthermore, mononucleated myotubes were also observed during primary myogenesis in amniotes. The mononucleated state of myogenic cells could be considered as an old phylogenetic, plesiomorphic feature, whereas direct multinuclearity of myotubes has a synapomorphic character. On the other hand, the explanation of this phenomenon could also be linked to the environmental conditions in which animals develop. The similarities observed in vertebrate myogenesis might result from a conservative myogenic programme governed by the Pax3/Pax7 and myogenic regulatory factor (MRF) network, whereas differences in anamniotes and amniotes are established by spatiotemporal pattern expression of MRFs during muscle differentiation and/or environmental conditions.
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http://dx.doi.org/10.1016/j.semcdb.2019.10.009DOI Listing
August 2020

Primary myogenesis in the sand lizard (Lacerta agilis) limb bud.

Dev Genes Evol 2019 11 18;229(5-6):147-159. Epub 2019 Jun 18.

Institute of Experimental Biology, Department of Animal Developmental Biology, University of Wroclaw, Sienkiewicza 21, 50-335, Wroclaw, Poland.

Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks.
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http://dx.doi.org/10.1007/s00427-019-00635-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6867991PMC
November 2019

Emerin Is Required for Proper Nucleus Reassembly after Mitosis: Implications for New Pathogenetic Mechanisms for Laminopathies Detected in EDMD1 Patients.

Cells 2019 03 13;8(3). Epub 2019 Mar 13.

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wroclaw, Fryderyka Joliot-Curie 14a, 50-383 Wroclaw, Poland.

Emerin is an essential LEM (LAP2, Emerin, MAN1) domain protein in metazoans and an integral membrane protein associated with inner and outer nuclear membranes. Mutations in the human gene coding for emerin result in the rare genetic disorder: Emery⁻Dreifuss muscular dystrophy type 1 (EDMD1). This disease belongs to a broader group called laminopathies-a heterogeneous group of rare genetic disorders affecting tissues of mesodermal origin. EDMD1 phenotype is characterized by progressive muscle wasting, contractures of the elbow and Achilles tendons, and cardiac conduction defects. Emerin is involved in many cellular and intranuclear processes through interactions with several partners: lamins; barrier-to-autointegration factor (BAF), β-catenin, actin, and tubulin. Our study demonstrates the presence of the emerin fraction which associates with mitotic spindle microtubules and centrosomes during mitosis and colocalizes during early mitosis with lamin A/C, BAF, and membranes at the mitotic spindle. Transfection studies with cells expressing EGFP-emerin protein demonstrate that the emerin fusion protein fraction also localizes to centrosomes and mitotic spindle microtubules during mitosis. Transient expression of emerin deletion mutants revealed that the resulting phenotypes vary and are mutant dependent. The most frequent phenotypes include aberrant nuclear shape, tubulin network mislocalization, aberrant mitosis, and mislocalization of centrosomes. Emerin deletion mutants demonstrated different chromatin binding capacities in an in vitro nuclear assembly assay and chromatin-binding properties correlated with the strength of phenotypic alteration in transfected cells. Aberrant tubulin staining and microtubule network phenotype appearance depended on the presence of the tubulin binding region in the expressed deletion mutants. We believe that the association with tubulin might help to "deliver" emerin and associated membranes to decondensing chromatin. Preliminary analyses of cells from Polish patients with EDMD1 revealed that for several mutations thought to be null for emerin protein, a truncated emerin protein was present. We infer that the EDMD1 phenotype may be strengthened by the toxicity of truncated emerin expressed in patients with certain nonsense mutations in .
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http://dx.doi.org/10.3390/cells8030240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468536PMC
March 2019

Folate-directed zinc (II) phthalocyanine loaded polymeric micelles engineered to generate reactive oxygen species for efficacious photodynamic therapy of cancer.

Photodiagnosis Photodyn Ther 2019 Mar 12;25:480-491. Epub 2019 Feb 12.

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wroclaw, Poland. Electronic address:

Targeted and effective drug transport is becoming an attractive option in cancer therapy since it can improve drug efficacy and reduce drugs' side effects in normal tissues. In addition to using specific surface ligand molecules, the selective drug delivery can be accomplished via enhanced permeability and retention effect. Therefore, in our studies, we entrapped zinc (II) phthalocyanine (ZnPc) - a second generation photosensitizer - in folate-functionalized micelles of the biocompatible, FDA-approved for biomedical application diblock copolymer methoxypoly(ethylene oxide)-b-poly(L-lactide) (mPEG-b-PLLA) and its derivative with folate (FA) attached to the end of PEG chain (FA-PEG-b-PLLA). Dynamic light scattering (DLS) measurements confirmed the micellar size to be <150 nm in diameter, a low polydispersity index, and good colloid stability of the studied nanocarriers, while atomic force microscopy (AFM) was used to study their morphology. The application potential of the resulting micelles was evaluated in cyto- and photocytotoxicity studies in conjunction with intracellular distribution and accumulation imaging of the photosensitizer delivered to ovarian carcinoma (SKOV-3) and metastatic melanoma (Me45) cell lines. Reactive oxygen species generation study was performed after photodynamic reaction, and cellular cytoskeleton reorganization was visualized after undergoing a photodynamic reaction. The results demonstrated that the functionalized polymeric micelles are promising nanocarriers for photodynamic therapy procedures and can be used in anticancer drug delivery.
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http://dx.doi.org/10.1016/j.pdpdt.2019.02.014DOI Listing
March 2019

Developmental Expression and Functions of the Small Heat Shock Proteins in .

Int J Mol Sci 2018 Nov 2;19(11). Epub 2018 Nov 2.

GReD, INSERM U1103, CNRS UMR6293, University of Clermont Auvergne, 28, Place Henri Dunant, 63000 Clermont-Ferrand, France.

Heat shock proteins (Hsps) form a large family of evolutionarily conserved molecular chaperones that help balance protein folding and protect cells from various stress conditions. However, there is growing evidence that Hsps may also play an active role in developmental processes. Here, we take the example of developmental expression and function of one class of Hsps characterized by low molecular weight, the small Hsps (sHsps). We discuss recent reports and genome-wide datasets that support vital sHsps functions in the developing nervous system, reproductive system, and muscles. This tissue- and time-specific sHsp expression is developmentally regulated, so that the enhancer sequence of an sHsp gene expressed in developing muscle, in addition to stress-inducible elements, also carries binding sites for myogenic regulatory factors. One possible reason for sHsp genes to switch on during development and in non-stress conditions is to protect vital developing organs from environmental insults.
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http://dx.doi.org/10.3390/ijms19113441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274884PMC
November 2018

Drosophila Hsp67Bc hot-spot variants alter muscle structure and function.

Cell Mol Life Sci 2018 Dec 21;75(23):4341-4356. Epub 2018 Jul 21.

Department of Animal Developmental Biology, Institute of Experimental Biology, University of Wroclaw, Sienkiewicza 21, 50-335, Wrocław, Poland.

The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. Here, we report the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, we observed a different impact on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant.
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http://dx.doi.org/10.1007/s00018-018-2875-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208764PMC
December 2018

ANTICANCER ACTIVITY OF OAT β-GLUCAN IN COMBINATION WITH ELECTROPORATION ON HUMAN CANCER CELLS.

Acta Pol Pharm 2017 Mar;74(2):616-623

The currently available data suggest that natural products may exert significant cytotoxic and immunomodulatory effects. Plant-derived chemotherapeutic agents such as taxol, etoposide or vincristine, currently used in cancer therapy, are prominent examples in this regard. However, there is a need for new and nat- ural anticancer compounds with low or without toxicity to normal cells. One of the active compounds responsible for the immune effects is β-glucan derived from cereals, fungi, seaweeds, yeasts and bacteria. The recent data suggest that β-glucans are potent immunomodulators with anticancer properties. Antitumor properties of fungi and yeast derived β-glucans have been widely recognized, but those polysaccharides are mostly insoluble, creating several problems especially in topical formulation. To overcome the issue of low water solubility, in the current study a more soluble β-glucan type from oats was chosen for the investigation of its antitumor activities. Cytotoxic effects were studied using a human melanoma cell line (Me45). The effect of electroporation on the antitumor activity of oat β-glucan was investigated as well. Cellular viability assessment, immuno-cytochemistry and immunofluochemistry were employed to evaluate biologic effects. Our results indicate strong anticancer properties of oat β-glucan, enhanced by electroporation.
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March 2017

Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function.

Int J Mol Sci 2016 Nov 19;17(11). Epub 2016 Nov 19.

GReD-Genetics, Reproduction and Development Laboratory, INSERM U1103, CNRS UMR6293, University of Clermont-Auvergne, 28 Place Henri-Dunant, 63000 Clermont-Ferrand, France.

The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To avoid or minimize the uncontrollable consequences of using the newly developed compounds, researchers seek a quick and effective means of their evaluation. In achieving this goal, the zebrafish () has proven to be a highly useful tool, mostly because of its fast growth and development, as well as the ability to absorb the molecules diluted in water through its skin and gills. In this review, we focus on the reports concerning the application of zebrafish as a model for assessing the impact of toxicants on skeletal muscles, which share many structural and functional similarities among vertebrates, including zebrafish and humans.
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http://dx.doi.org/10.3390/ijms17111941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133936PMC
November 2016

Does the grass snake (Natrix natrix) (Squamata: Serpentes: Natricinae) fit the amniotes-specific model of myogenesis?

Protoplasma 2017 Jul 10;254(4):1507-1516. Epub 2016 Nov 10.

Department of Animal Developmental Biology, Institute of Experimental Biology, University of Wroclaw, Sienkiewicza 21, 50-335, Wroclaw, Poland.

In the grass snake (Natrix natrix), the newly developed somites form vesicles that are located on both sides of the neural tube. The walls of the vesicles are composed of tightly connected epithelial cells surrounding the cavity (the somitocoel). Also, in the newly formed somites, the Pax3 protein can be observed in the somite wall cells. Subsequently, the somite splits into three compartments: the sclerotome, dermomyotome (with the dorsomedial [DM] and the ventrolateral [VL] lips) and the myotome. At this stage, the Pax3 protein is detected in both the DM and VL lips of the dermomyotome and in the mononucleated cells of the myotome, whereas the Pax7 protein is observed in the medial part of the dermomyotome and in some of the mononucleated cells of the myotome. The mononucleated cells then become elongated and form myotubes. As myogenesis proceeds, the myotome is filled with multinucleated myotubes accompanied by mononucleated, Pax7-positive cells (satellite cells) that are involved in muscle growth. The Pax3-positive progenitor muscle cells are no longer observed. Moreover, we have observed unique features in the differentiation of the muscles in these snakes. Specifically, our studies have revealed the presence of two classes of muscles in the myotomes. The first class is characterised by fast muscle fibres, with myofibrils equally distributed throughout the sarcoplasm. In the second class, composed of slow muscle fibres, the sarcoplasm is filled with lipid droplets. We assume that their storage could play a crucial role during hibernation in the adult snakes. We suggest that the model of myotomal myogenesis in reptiles, birds and mammals shows the same morphological and molecular character. We therefore believe that the grass snake, in spite of the unique features of its myogenesis, fits into the amniotes-specific model of trunk muscle development.
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http://dx.doi.org/10.1007/s00709-016-1040-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487930PMC
July 2017

The Effect of Millisecond Pulsed Electric Fields (msPEF) on Intracellular Drug Transport with Negatively Charged Large Nanocarriers Made of Solid Lipid Nanoparticles (SLN): In Vitro Study.

J Membr Biol 2016 10 12;249(5):645-661. Epub 2016 May 12.

Department of Biomedical Engineering, Faculty of Fundamental Problems of Technology, Wroclaw University of Science and Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wroclaw, Poland.

Drug delivery technology is still a dynamically developing field of medicine. The main direction in nanotechnology research (nanocarriers, nanovehicles, etc.) is efficient drug delivery to target cells with simultaneous drug reduction concentration. However, nanotechnology trends in reducing the carrier sizes to several nanometers limit the volume of the loaded substance and may pose a danger of uncontrolled access into the cells. On the other hand, nanoparticles larger than 200 nm in diameter have difficulties to undergo rapid diffusional transport through cell membranes. The main advantage of large nanoparticles is higher drug encapsulation efficiency and the ability to deliver a wider array of drugs. Our present study contributes a new approach with large Tween 80 solid lipid nanoparticles SLN (i.e., hydrodynamic GM-SLN-glycerol monostearate, GM, as the lipid and ATO5-SLNs-glyceryl palmitostearate, ATO5, as the lipid) with diameters DH of 379.4 nm and 547 nm, respectively. They are used as drug carriers alone and in combination with electroporation (EP) induced by millisecond pulsed electric fields. We evaluate if EP can support the transport of large nanocarriers into cells. The study was performed with two cell lines: human colon adenocarcinoma LoVo and hamster ovarian fibroblastoid CHO-K1 with coumarin 6 (C6) as a fluorescent marker for encapsulation. The biological safety of the potential treatment procedure was evaluated with cell viability after their exposure to nanoparticles and EP. The EP efficacy was evaluated by FACS method. The impact on intracellular structure organization of cytoskeleton was visualized by CLSM method with alpha-actin and beta-tubulin. The obtained results indicate low cytotoxicity of both carrier types, free and loaded with C6. The evaluation of cytoskeleton proteins indicated no intracellular structure damage. The intracellular uptake and accumulation show that SLNs do not support transport of C6 coumarin. Only application of electroporation improved the transport of encapsulated and free C6 into both treated cell lines.
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http://dx.doi.org/10.1007/s00232-016-9906-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045845PMC
October 2016

Preparation and characterization of new zinc(II) phthalocyanine - Containing poly(l-lactide)-b-poly(ethylene glycol) copolymer micelles for photodynamic therapy.

J Photochem Photobiol B 2016 Jul 16;160:185-97. Epub 2016 Apr 16.

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland. Electronic address:

Poly(l-lactide)-b-poly(ethylene oxide) block copolymer (mPEG-b-PLLA) micelles were fabricated and applied as a new biodegradable and biocompatible nanocarrier for solubilization of hydrophobic zinc (II) phthalocyanine (ZnPc). The nanocarrier demonstrated a good colloidal stability and its in vitro sustained cargo release profile was assessed. Photobleaching of ZnPc, both in its native form and encapsulated in the obtained polymeric micelles, was studied by means of spectroscopic measurements. The photodynamic reaction (PDR) protocol for cyto- and photocytotoxicity was performed on metastatic melanoma cells (Me45), normal human keratinocytes (HaCaT) being used for comparison. The intracellular accumulation of free and encapsulated ZnPc was visualized at various time periods (1, 3 and 24h). The proapoptotic potential of the encapsulated phthalocyanine was evaluated by monitoring DNA double strand break damage fragmentation (TUNEL assay) and caspase 3/7 activity. In addition, in vitro biocompatibility studies were conducted by determining hemolytic activity of Zn-Pc-loaded mPEG-b-PLLA micelles and their lack of cytotoxicity against macrophages (P388/D1) and endothelial cells (HUV-EC-C). Our results suggest that the PDR using Zn-Pc-loaded mPEG-b-PLLA micelles can be effective in inhibiting tumor cell growth and apoptosis induction with higher responses, observed for Me45 cells. Additionally, the ZnPc-loaded micelles appear to be hemato-biocompatible and safe for normal keratinocytes, macrophages and endothelial cells.
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http://dx.doi.org/10.1016/j.jphotobiol.2016.04.018DOI Listing
July 2016

Electroporation and lipid nanoparticles with cyanine IR-780 and flavonoids as efficient vectors to enhanced drug delivery in colon cancer.

Bioelectrochemistry 2016 Aug 27;110:19-31. Epub 2016 Feb 27.

Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.

Nanocarriers and electroporation (also named electropermeabilization) are convenient methods to increase drug transport. In the current study, we present an effective support of drug delivery into cancer cells, utilizing these methods. We compare the efficiency of each of them and their combination. Multifunctional solid lipid nanoparticles (SLNs) loaded with a cyanine-type IR-780 - acting as a diagnostic agent and a photosensitizer, and a flavonoid derivative - baicalein (BAI) or fisetin (FIS) as a therapeutic cargo - were fabricated via solvent-diffusion method. A therapy supplemented with flavonoids may provide a more precise method to apply desirable lower drug doses and is more likely to result in lower toxicity and a decrease in tumor growth. The SLNs were stabilized with Phospholipon 90G at various concentrations; cetyl palmitate (CP) was applied as a solid matrix. The obtained nanosystems were characterized by dynamic light scattering (size along with size distribution), UV-vis (cargos encapsulation efficiency) and atomic force microscopy (morphology and shape). The obtained SLNs were used as drug carriers alone and in combination with electropermeabilization induced by millisecond pulsed electric fields of high intensity. Two cell lines were selected for the study: LoVo and CHO-K1. The viability was assessed after electroporation alone, the use of electroporation and nanoparticles, and nanoparticles or drugs alone. The intracellular accumulation of cyanine IR-780 and the impact on intracellular structure organization of cytoskeleton was visualized with confocal microscopy method with alpha-actin and beta-tubulin. In this study, the efficacy of nanoparticles with mixed cargo, additionally enhanced by electroporation, is demonstrated to act as an anticancer modality to eliminate cancer cells.
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http://dx.doi.org/10.1016/j.bioelechem.2016.02.013DOI Listing
August 2016

Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

Protoplasma 2016 May 25;253(3):943-956. Epub 2015 Jul 25.

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, Joliot- Curie 14a, 50-383, Wrocław, Poland.

Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.
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http://dx.doi.org/10.1007/s00709-015-0861-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819936PMC
May 2016

Unique features of myogenesis in Egyptian cobra (Naja haje) (Squamata: Serpentes: Elapidae).

Protoplasma 2016 Mar 30;253(2):625-33. Epub 2015 May 30.

Department of Animal Developmental Biology, Institute of Experimental Biology, University of Wrocław, 21 Sienkiewicza Str., 53-335, Wrocław, Poland.

During early stages of myotomal myogenesis, the myotome of Egyptian cobra (Naja haje) is composed of homogenous populations of mononucleated primary myotubes. At later developmental phase, primary myotubes are accompanied by closely adhering mononucleated cells. Based on localization and morphology, we assume that mononucleated cells share features with satellite cells involved in muscle growth. An indirect morphological evidence of the fusion of mononucleated cells with myotubes is the presence of numerous vesicles in the subsarcolemmal region of myotubes adjacent to mononucleated cell. As differentiation proceeded, secondary muscle fibres appeared with considerably smaller diameter as compared to primary muscle fibre. Studies on N. haje myotomal myogenesis revealed some unique features of muscle differentiation. TEM analysis showed in the N. haje myotomes two classes of muscle fibres. The first class was characterized by typical for fast muscle fibres regular distribution of myofibrils which fill the whole volume of muscle fibre sarcoplasm. White muscle fibres in studied species were a prominent group of muscles in the myotome. The second class showed tightly paced myofibrils surrounding the centrally located nucleus accompanied by numerous vesicles of different diameter. The sarcoplasm of these cells was characterized by numerous lipid droplets. Based on morphological features, we believe that muscle capable of lipid storage belong to slow muscle fibres and the presence of lipid droplets in the sarcoplasm of these muscles during myogenesis might be a crucial adaptive mechanisms for subsequent hibernation in adults. This phenomenon was, for the first time, described in studies on N. haje myogenesis.
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http://dx.doi.org/10.1007/s00709-015-0840-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783446PMC
March 2016

Oxidative modification induced by photodynamic therapy with Photofrin®II and 2-methoxyestradiol in human ovarian clear carcinoma (OvBH-1) and human breast adenocarcinoma (MCF-7) cells.

Biomed Pharmacother 2015 Apr 19;71:30-6. Epub 2015 Feb 19.

Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wrocław, Poland. Electronic address:

Ovarian cancer is among the most lethal cancers in women. The successful anticancer treatment depends on the effectiveness of cytotoxic effect of applied therapeutic procedures either alone or in combination with other treatments. Photodynamic therapy (PDT) is a relatively new method of anticancer therapy. Its dominant mechanism of action is the over-production of reactive oxygen species induced by oxidative stress in malignant cells, which attack lipid membranes, proteins and nucleic acids. One of the important mechanisms is induction of unfolded protein response, ubiquitin-proteasome pathway of protein degradation. The aim of this study was to evaluate the cytotoxic effect of various protective enzymes in ovarian carcinoma clear cell line in comparison to the model breast cell line after photodynamic reaction and photodynamic reaction with 2-methoxyestradiol (2-Me). Human malignant ovarian cell line (OvBH-1) was used and human breast adenocarcinoma cells (MCF-7) were used as a control. Photodynamic reaction (PDR) with Photofrin(®)II and Ph(®)II with 2-Me was performed. The expression of protective proteins by immunocytochemistry (HSP70 and iNOS) and western blot (Hsp27 and Hsp70) methods was evaluated directly, 3 and 6 h after PDR. The changes in cells' cytoskeleton were evaluated using immunofluorescence by confocal microscopy. The expression of iNOS was observed for both experiments with differential intensity and quantity. A higher expression of Hsp70 in MCF-7 cells was observed than in OvBh-1 cells. The reorganization of cytoskeleton and nucleus was observed after 3 and 6 h after exposition to light.
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http://dx.doi.org/10.1016/j.biopha.2015.02.008DOI Listing
April 2015

The photodynamic effect of far-red range phthalocyanines (AlPc and Pc green) supported by electropermeabilization in human gastric adenocarcinoma cells of sensitive and resistant type.

Biomed Pharmacother 2015 Feb 24;69:145-52. Epub 2014 Nov 24.

Institute of Pathology, Charité Universitätsmedizin, Berlin, Germany.

Introduction: Electroporation (EP) is commonly applied for effective drug transport thorough cell membranes based on the application of electromagnetic field. When applied with cytostatics, it is called electrochemotherapy (ECT) - a quite new method of cancer treatment. A high-voltage pulse causes the formation of temporary pores in the cell membrane which create an additional way for the intracellular drug transport. In the current work, EP was effectively merged with the already known photodynamic therapy (PDT) to selective photosensitizers' delivery to diseased tissue. The application of electroporation can reduce the dose of applied drug.

Research Objective: The aim of research was to evaluate the effectiveness of photodynamic reaction using two near infrared cyanines (AlPc and Pc green) combined with electroporation in two human gastric adenocarcinoma cell lines.

Materials And Methods: Two human cell lines - EPG85-257P (parental) and EPG85-257RDB (resistant to daunorubicin) - of gastric cancer were used. The effect of two photosensitizers (aluminum 1,8,15,22-tetrakis(-phenylthio)-29H,31H-phthalocyanine chloride and Phthalocyanine green) was investigated. The efficiency of EP parameters was assessed by propidium iodide uptake. The viability assay was applied to analyse EP, PDT and EP-PDT effect. Cyanine localization was determined by confocal microscopy. Immunocytochemical evaluation of manganese superoxide dismutase and glutathione S-transferase-pi was determined after applied therapies.

Results: PDT in combination with EP affected the viability of EPG85-257P and EPG85-257RDB cells negatively while both cyanine were used. The most evident changes were observed in the following concentrations: 15, 10 and 5μM. The optimal field strength for enhanced EP-PDT was 800 and 1200V/cm. AlPc distributed selectively in the lysosomes of parental cell line.

Conclusions: PDT, enhanced by EP, caused decreased viability when compared to the application of PDT alone. Both phthalocyanines found to be more effective after electroporation. Due to the low concentration of light-sensitive compounds and safety of electroporation itself, a treatment plan can be an alternative therapeutic modality against gastric adenocarcinomas.
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http://dx.doi.org/10.1016/j.biopha.2014.11.017DOI Listing
February 2015

Doxorubicin delivery enhanced by electroporation to gastrointestinal adenocarcinoma cells with P-gp overexpression.

Bioelectrochemistry 2014 Dec 4;100:96-104. Epub 2014 Apr 4.

Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-367 Wroclaw, Poland.

Electroporation (EP) can effectively support the penetration of macromolecules from the extracellular space into cells. Electropores induced by the influence of electromagnetic field generate additional paths of transport for macromolecules. The aim of this study was evaluation of the electroporation effect on doxorubicin transport efficiency to human colon (LoVo and LoVo/DX) and gastric (EPG85-257/P and EPG85-257/RDB) adenocarcinoma cells with overexpression of P-glycoprotein and murine macrophage cell line (P388/D1). In our EP experiments cells were placed into a cuvette with aluminum electrodes and pulsed with five square electric pulses of 1300 V/cm and duration of 50 μs each. Cells were also treated with low doxorubicin concentration ([DOX]=1.7 μM). The ultrastructure (TEM) and changes of P-glycoprotein expression of tumor cells subjected to electric field were monitored. The mitochondrial cell function and trypan blue staining were evaluated after 24h. Our results indicate the most pronounced effect of EP with DOX and disturbed ultrastructure in resistant gastric and colon cells with decrease of P-gp expression. Electroporation may be an attractive delivery method of cytostatic drugs in chemotherapy, enabling reduction of drug dose, exposure time and side effects.
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http://dx.doi.org/10.1016/j.bioelechem.2014.03.013DOI Listing
December 2014

The influence of retraction agents on cytoskeleton reorganization and oxidative stress in primary human gingival fibroblasts (HGFs).

Arch Oral Biol 2014 Mar 8;59(3):341-8. Epub 2014 Jan 8.

Department of Medical Biochemistry, Wroclaw Medical University, Poland. Electronic address:

Objective: Contemporary gingival retraction chemicals are not without disagreeable side-effects; there appears to be no best gingival retraction agent. The aim of this research was to select the most biocompatible retraction agents based on examination of the parameters of oxidative stress in fibroblasts derived from human primary cell culture.

Design: In this in vitro study we evaluated parameters of oxidative stress after treatment with retraction agents. Visine, Afrin, Neosynephrin, Strazolin and Adrenaline were the commercial products studied as gingival retraction agents. Additionally we examined three experimental agents. We determined lipid peroxidation and protein damage and monitored changes in cellular cytoskeleton proteins. Proliferative and survival efficiency were also evaluated.

Results: Oxidative changes included by evaluated retraction agents were at the lowest level in the case of the experimental gels. Also cytoskeleton observations suggest that the experimental agents did not degrade the cellular structure of human gingival fibroblasts (HGFs).

Conclusions: The current study was performed because of a need to project new nontoxic and save retraction agents for peridontological therapeutic usage. We suggest that the new investigational gels are most biocompatible with periodontal tissues and can be applied as new vasoconstrictor chemical retraction agents.
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http://dx.doi.org/10.1016/j.archoralbio.2013.12.011DOI Listing
March 2014

Contribution of small heat shock proteins to muscle development and function.

FEBS Lett 2014 Feb 17;588(4):517-30. Epub 2014 Jan 17.

Department of Animal Developmental Biology, University of Wroclaw, 21 Sienkiewicza Street, 50-335 Wroclaw, Poland. Electronic address:

Investigations undertaken over the past years have led scientists to introduce the concept of protein quality control (PQC) systems, which are responsible for polypeptide processing. The PQC system monitors proteostasis and involves activity of different chaperones such as small heat shock proteins (sHSPs). These proteins act during normal conditions as housekeeping proteins regulating cellular processes, and during stress conditions. They also mediate the removal of toxic misfolded polypeptides and thereby prevent development of pathogenic states. It is postulated that sHSPs are involved in muscle development. They could act via modulation of myogenesis or by maintenance of the structural integrity of signaling complexes. Moreover, mutations in genes coding for sHSPs lead to pathological states affecting muscular tissue functioning. This review focuses on the question how sHSPs, still relatively poorly understood proteins, contribute to the development and function of three types of muscle tissue: skeletal, cardiac and smooth.
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http://dx.doi.org/10.1016/j.febslet.2014.01.005DOI Listing
February 2014

Muscle development, regeneration and laminopathies: how lamins or lamina-associated proteins can contribute to muscle development, regeneration and disease.

Cell Mol Life Sci 2013 Aug 10;70(15):2713-41. Epub 2012 Nov 10.

Department of Animal Developmental Biology, University of Wroclaw, 21 Sienkiewicza Street, 50-335, Wroclaw, Poland.

The aim of this review article is to evaluate the current knowledge on associations between muscle formation and regeneration and components of the nuclear lamina. Lamins and their partners have become particularly intriguing objects of scientific interest since it has been observed that mutations in genes coding for these proteins lead to a wide range of diseases called laminopathies. For over the last 10 years, various laboratories worldwide have tried to explain the pathogenesis of these rare disorders. Analyses of the distinct aspects of laminopathies resulted in formulation of different hypotheses regarding the mechanisms of the development of these diseases. In the light of recent discoveries, A-type lamins--the main building blocks of the nuclear lamina--together with other key elements, such as emerin, LAP2α and nesprins, seem to be of great importance in the modulation of various signaling pathways responsible for cellular differentiation and proliferation.
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http://dx.doi.org/10.1007/s00018-012-1190-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3708280PMC
August 2013

Reptilian myotomal myogenesis-lessons from the sand lizard Lacerta agilis L. (Reptilia, Lacertidae)Update.

Zoology (Jena) 2012 Oct 18;115(5):330-8. Epub 2012 Aug 18.

Department of Animal Histology and Embryology, Silesian University, 9 Bankowa Str., 40-007 Katowice, Poland.

Reptilian myotomal myogenesis is poorly understood. This paper reports on structural, ultrastructural and immunocytochemical studies of muscle differentiation in sand lizard (Lacerta agilis) embryos. During somitogenesis, the somites are composed of epithelial vesicles with a centrally located somitocoel. At later developmental stages the ventral portion of the somite cortex disaggregates into the sclerotome mesenchyme, while the dorsal wall of the somite differentiates into dermomyotome. At these developmental stages, mononucleated cells of the dermomyotome are Pax3-positive. The dermomyotome layer forms the dorsomedial and ventromedial lips. The myotome is first composed of mono- and then of multinucleated myotubes and small mononucleated cells that occur in the vicinity of the myotubes. These mononucleated cells exhibit low proliferative potential as revealed by the use of PCNA antibody. At subsequent stages of myogenesis the mononucleated cells express Pax7 protein, a marker of satellite cells, and assume ultrastructural features characteristic of satellite cells. Some of the mononucleated cells contribute to muscle growth, being involved in fusion with differentiating muscle fibers. This study revealed similarities of myotomal myogenesis in reptiles to that of other vertebrates.
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http://dx.doi.org/10.1016/j.zool.2012.04.002DOI Listing
October 2012

The different function of single phosphorylation sites of Drosophila melanogaster lamin Dm and lamin C.

PLoS One 2012 29;7(2):e32649. Epub 2012 Feb 29.

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.

Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S(25)E, S(45)E, T(435)E, S(595)E). We also analyzed lamin C (A-type) and its mutant S(37)E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R(64)H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S(45)E mutant was insoluble, in contrast to lamin C S(37)E. Lamin Dm T(435)E (C-terminal cdc2 site) and R(64)H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S(45)E and T(435)E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T(435)E was cytoplasmic and showed higher mobility in FRAP assay.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290585PMC
July 2012

Identification of new in vivo phosphosites on lamin Dm - the evidence of heterogeneity of phosphorylation sites in different Drosophila tissues.

Nucleus 2011 Sep-Oct;2(5):478-88. Epub 2011 Sep 1.

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, Przybyszewskiego St. 63/78, 51-148 Wrocław, Poland.

Changes in the nuclear structure and function during the cell cycle are thought to be correlated with lamins phosphorylation. Here, we report the identification of new in vivo phosphorylation sites on Drosophila melanogater lamin Dm using immunoisolation and mass spectrometry with collision-induced peptide fragmentation (Electrospray-Linear Trap Quadrupole- Fourier Transform Ion Cyclotron Resonance MS/MS). We identified S19 and confirmed previously suggested S595 as phosphorylated amino acid residues on embryonic lamin Dm. We also found that T597 is phosphorylated in vivo in cultured Kc cells while S595 in embryos, which suggests that different neighboring phosphoacceptors may be modified within the same region. We demonstrate also that Drosophila melanogaster lamin Dm in very early (syncytial) embryos is almost completely dispersed through the entire embryo. Only fraction of lamin Dm is associated with nuclei and nuclear envelopes. In later stages, due to the synchronization of mitosis, lamin Dm may be both nuclear and cytoplasmic in the same embryo. Our results provide a new and essential data for better understanding of the lamin phosporylation in development and cell cycle regulation in Drosophila.
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http://dx.doi.org/10.4161/nucl.2.5.17864DOI Listing
March 2012

Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope.

Cell Tissue Res 2011 Apr 24;344(1):97-110. Epub 2011 Feb 24.

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63/77, 51-148, Wrocław, Poland.

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2γ, which was downregulated during development similarly to XLAP2ω. Embryonic isoforms XLAP2ω and XLAP2γ were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2β were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2β was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2β was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains.
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http://dx.doi.org/10.1007/s00441-011-1129-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112025PMC
April 2011

Laminopathies: the molecular background of the disease and the prospects for its treatment.

Cell Mol Biol Lett 2011 Mar 27;16(1):114-48. Epub 2010 Dec 27.

Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-148, Wroclaw, Poland.

Laminopathies are rare human degenerative disorders with a wide spectrum of clinical phenotypes, associated with defects in the main protein components of the nuclear envelope, mostly in the lamins. They include systemic disorders and tissue-restricted diseases. Scientists have been trying to explain the pathogenesis of laminopathies and find an efficient method for treatment for many years. In this review, we discuss the current state of knowledge about laminopathies, the molecular mechanisms behind the development of particular phenotypes, and the prospects for stem cell and/or gene therapy treatments.
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http://dx.doi.org/10.2478/s11658-010-0038-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275778PMC
March 2011

[Karyoskeletal and nuclear envelope proteins in cell cycle progression--known proteins in new functions].

Postepy Biochem 2010 ;56(4):362-72

Pracownia Białek Jadrowych, Wydział Biotechnologii, Uniwersytet Wrocławski, Wrocław.

The cell nucleus is separated from a cytoplasm by a nuclear envelope (NE) composed of nuclear lamina (NL), outer (ONM) and inner nuclear membrane (INM), connected in the region of nuclear pore complexes (NPC), which are sites for macromolecular transport between the nucleus and the cytoplasm. The nuclear lamina is an essential structure mainly composed of type V intermediate filament proteins, A- and B-type lamins, located between the inner nuclear membrane and the peripheral chromatin. Nuclear envelope, which is composed of integral membrane proteins of the INM (LAP1, LAP2, emerin, MAN1, LBR), has many functions including: connection of nucleoskeleton with cytoskeleton, nuclear lamina meshwork and chromatin. This structure plays a role in maintenance of nuclear shape, spacing of nuclear pore complexes, organization of heterochromatin, DNA replication, and regulation of transcription factors. During cell division NE undergoes depolimerization and reassociation. Latest data suggests, that proteins creating nuclear envelope take part in mitosis.
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September 2011