Publications by authors named "Malgorzata Daczewska"

32 Publications

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

Differentiation of skeletal muscles.

Semin Cell Dev Biol 2020 08 4;104:1-2. Epub 2020 Jun 4.

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

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http://dx.doi.org/10.1016/j.semcdb.2020.05.008DOI Listing
August 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

The effect of muscle glycogen phosphorylase (Pygm) knockdown on zebrafish morphology.

Int J Biochem Cell Biol 2020 01 17;118:105658. Epub 2019 Nov 17.

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

Muscle glycogen phosphorylase (PYGM) is a key enzyme in the first step of glycogenolysis. Mutation in the PYGM gene leads to autosomal recessive McArdle disease. Patients suffer from exercise intolerance with premature fatigue, muscle cramps and myalgia due to lack of available glucose in muscles. So far, no efficient treatment has been found. The zebrafish has many experimental advantages, and was successfully implemented as an animal model of human myopathies. Since zebrafish skeletal muscles share high similarity with human skeletal muscles, it is our animal of choice to investigate the impact of Pygm knockdown on skeletal muscle tissue. The two forms of the zebrafish enzyme, Pygma and Pygmb, share more than 80% amino acid sequence identity with human PYGM. We show that the Pygm level varies at both the mRNA and protein level in distinct stages of zebrafish development, which is correlated with glycogen level. The Pygm distribution in muscles varies from dispersed to highly organized at 72 hpf. The pygma and pygmb morpholino knockdown resulted in a reduced Pygm level in zebrafish morphants, which exhibited altered, disintegrated muscle structure and accumulation of glycogen granules in the subsarcolemmal region. Thus, lowering the Pygm level in zebrafish larvae leads to an elevated glycogen level and to morphological muscle changes mimicking the symptoms of human McArdle disease. The zebrafish model of this human disease might contribute to further understanding of its molecular mechanisms and to the development of appropriate treatment.
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http://dx.doi.org/10.1016/j.biocel.2019.105658DOI Listing
January 2020

Correction: Calcium electroporation for treatment of sarcoma in preclinical studies.

Oncotarget 2019 Aug 20;10(49):5118. Epub 2019 Aug 20.

Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland.

[This corrects the article DOI: 10.18632/oncotarget.24352.].
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http://dx.doi.org/10.18632/oncotarget.27158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707949PMC
August 2019

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

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

Calcium electroporation for treatment of sarcoma in preclinical studies.

Oncotarget 2018 Feb 30;9(14):11604-11618. Epub 2018 Jan 30.

Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland.

Calcium electroporation (CaEP) describes the use of electric pulses (electroporation) to transiently permeabilize cells to allow supraphysiological doses of calcium to enter the cytosol. Calcium electroporation has successfully been investigated for treatment of cutaneous metastases in a clinical study. This preclinical study explores the possible use of calcium electroporation for treatment of sarcoma. A normal murine muscle cell line (C2C12), and a human rhabdomyosarcoma cell line (RD) were used in the undifferentiated and differentiated state. Electroporation was performed using 8 pulses of 100 μs at 600-1000 V/cm; with calcium (0, 0.5, 1, and 5 mM). Viability was examined by MTS assay, intracellular calcium levels were measured, and expression of plasma membrane calcium ATPase (PMCA) was investigated using western blotting. Calcium/sodium exchanger (NCX1), ryanodine receptor (RyR1) expression and cytoskeleton structure (zyxin/actin) were assessed by immunofluorescence. CaEP efficiency on RD tumors was tested in immuno-deficient mice. CaEP was significantly more efficient in RD than in normal cells. Intracellular Ca levels after CaEP increased significantly in RD, whereas a lower increase was seen in normal cells. CaEP caused decreased expression of PMCA and NCX1 in malignant cells and RyR1 in both cell lines whereas normal cells exhibited increased expression of NCX1 after CaEP. Calcium electroporation also affected cytoskeleton structure in malignant cells. This study showed that calcium electroporation is tolerated significantly better in normal muscle cells than sarcoma cells and as an inexpensive and simple cancer treatment this could potentially be used in connection with sarcoma surgery for local treatment.
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http://dx.doi.org/10.18632/oncotarget.24352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837766PMC
February 2018

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

Applications of calcium electroporation to effective apoptosis induction in fibrosarcoma cells and stimulation of normal muscle cells.

Bioelectrochemistry 2016 Jun 27;109:70-8. Epub 2016 Jan 27.

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

The electroporation (EP) supports various types of anticancer therapies by the selective transport of cytostatics. Increase in intracellular calcium level by EP may be a new approach to fibrosarcoma treatment. Calcium is one of the most important factors of cell proliferation, differentiation and cell death (apoptosis or necrosis). Calcium level balanced by electroporation can cause different effects on normal and pathological cells. The efficiency and safety of electroporation combined with Ca(2+) ions were examined in our study. The two muscle cell lines were used: normal rat skeletal muscle cells - L6 and cancer muscle cells - Wehi-164 (fibrosarcoma). Two CaCl2 concentrations were tested: 0.5 mM and 5 mM combined with EP parameters: 1000 V/cm, 1200 V/cm, and 1500 V/cm. The results show that EP supported by Ca(2+) is cytotoxic for Wehi-164 cells and simultaneously safe for normal muscle cells. The main type of cell death - apoptosis - was confirmed by Tunnel and Annexin V/PI assay. Additionally, sPLA2 pro-tumorigenic influence was proved by immunocytochemistry. Moreover, EP with 0.5 mM of Ca(2+) slightly stimulates the normal muscle cells - L6 to increase proliferation.
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http://dx.doi.org/10.1016/j.bioelechem.2016.01.005DOI Listing
June 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

Model organisms in the fight against muscular dystrophy: lessons from drosophila and Zebrafish.

Molecules 2015 Apr 9;20(4):6237-53. Epub 2015 Apr 9.

GReD (Genetics, Reproduction and Development laboratory), INSERM U1103, CNRS UMR6293, University of Clermont-Ferrand, 28 place Henri-Dunant, 63000 Clermont-Ferrand, France.

Muscular dystrophies (MD) are a heterogeneous group of genetic disorders that cause muscle weakness, abnormal contractions and muscle wasting, often leading to premature death. More than 30 types of MD have been described so far; those most thoroughly studied are Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1) and congenital MDs. Structurally, physiologically and biochemically, MDs affect different types of muscles and cause individual symptoms such that genetic and molecular pathways underlying their pathogenesis thus remain poorly understood. To improve our knowledge of how MD-caused muscle defects arise and to find efficacious therapeutic treatments, different animal models have been generated and applied. Among these, simple non-mammalian Drosophila and zebrafish models have proved most useful. This review discusses how zebrafish and Drosophila MD have helped to identify genetic determinants of MDs and design innovative therapeutic strategies with a special focus on DMD, DM1 and congenital MDs.
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http://dx.doi.org/10.3390/molecules20046237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272363PMC
April 2015

Drosophila small heat shock protein CryAB ensures structural integrity of developing muscles, and proper muscle and heart performance.

Development 2015 Mar;142(5):994-1005

GReD - INSERM U1103, CNRS UMR6293, Clermont Université, 28, place Henri Dunant, Clermont-Ferrand 63000, France

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.
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http://dx.doi.org/10.1242/dev.115352DOI Listing
March 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

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

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

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells.

Asian Pac J Trop Biomed 2011 Apr;1(2):94-8

Department of Medical Biochemistry, Medical University, Chalubinskiego 10 St., 50-368 Wroclaw, Poland.

Objective: To estimate electroporation (EP) influence on malignant and normal cells.

Methods: Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following: 250, 1 000, 1 750, 2 500 V/cm; 50 µs by 5 impulses for every case. The viability of cells after EP was estimated by MTT assay. The ultrastructural analysis was observed by transmission electron microscope (Zeiss EM 900).

Results: In the current study we observed the intracellular effect following EP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP. Conversely, we showed that EP in some conditions can stimulate cells to proliferation. Some changes induced by EP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters of EP (250 and 1 000 V/cm). After applying higher electric field intensities (2 500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications after EP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.

Conclusions: We can claim that EP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.
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http://dx.doi.org/10.1016/S2221-1691(11)60003-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609180PMC
April 2011

The estimation of oxidative stress markers and apoptosis in right atrium auricles cardiomyocytes of patients undergoing surgical heart revascularisation with the use of warm blood cardioplegia.

Folia Histochem Cytobiol 2010 Jan;48(2):202-7

Department of Cardiac Surgery, Medical University of Wroclaw, Poland.

Oxidative stress markers and apoptosis were estimated during elective surgical heart revascularization. Eight patients with good ejection fraction underwent coronary artery bypass grafting (CABG) with the use of warm blood cardioplegia. Two right atrium auricle biopsy specimens were collected before and after the operation. Specimens underwent immunocytochemical analysis of mitochondrial manganese superoxide dismutase (MnSOD) expression and apoptosis estimation by the TUNEL method. Ultrastructure analysis under electron microscope was made. Satisfactory results of the operation were obtained. After CABG the MnSOD expression increase in sections of auricles was observed through the increase of stain intensity and the percentage of cells with positive stain (from 30 to 80%). The apoptotic cells percentage remained at approximately the same level. Under the electron microscope insignificant pathological changes were observed. On this basis one may assume that in the case of cardiosurgical procedures with short aorta cross-clamping time and low operation risk level the application of cardioplegia sufficiently prevents reactive oxygen forms (ROF) cytotoxic activity although it does not inhibit the expression of oxidative stress (OS) markers. In our opinion the method of examining right atrium sections is safe and provides results comparable with other publications. It may also be a voice in the discussion on new methods of heart protection during cardiac surgery procedures.
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http://dx.doi.org/10.2478/v10042-010-0012-4DOI Listing
January 2010

Muscle development and regeneration in normal and pathological conditions: learning from Drosophila.

Curr Pharm Des 2010 ;16(8):929-41

Zoology Dept, University of Wroclaw, 21, Sienkiewicza Street, 50-335 Wroclaw, Poland.

The recent demonstration that, throughout evolution, many molecular mechanisms have been highly conserved is fundamental to the advancement of our knowledge on muscle development and regeneration. Research has provided new insights into genetic cascades governing early steps of embryonic myogenesis and the regeneration of adult muscle in normal and pathological conditions, thus revealing significant similarity of both processes. Here we provide a current view on genetic mechanisms underlying muscle regeneration with a special focus on regeneration processes that take place in diseased and aging human muscle. Through examples of Drosophila models of human muscular diseases, we discuss potential impact they might have on uncovering molecular bases and identifying new treatments of muscle disorders. Taking advantage of evolutionarily conserved aspects of muscle development and the relative ease by which molecular pathways can be uncovered and dissected in a simple animal model, the fruit fly, we provide a comprehensive analysis of muscle development in Drosophila. Importantly, identification of muscle stem cell like adult muscle precursors in Drosophila makes fruit fly an attractive model system for studying muscle stem cell biology and muscle regeneration. In support of this assumption, recent studies in our laboratory provide arguments that important insights into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.
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http://dx.doi.org/10.2174/138161210790883462DOI Listing
June 2010

The Australian lungfish (Neoceratodus forsteri) - fish or amphibian pattern of muscle development?

Int J Dev Biol 2008 ;52(2-3):279-86

Department of General Zoology, Zoological Institute, University of Wroclaw, Wroclaw, Poland.

The Australian lungfish Neoceratodus forsteri (Dipnoi-Sarcoterygians) is a likely candidate for the extant sister group of Tetrapoda. Transmission electron and light microscopy analysis revealed that the arrangement of somite cells of the lungfish resembles the structure of the urodelan somite. On the other hand, the pattern of early muscle formation in N. forsteri is similar to that found in the Siberian sturgeon (Acipenser baeri). During the early stages of myogenesis of N. forsteri, somite-derived cells fuse to form multinucleated muscle lamellae. During later stages, mononucleated undifferentiated cells are first observed in the intermyotomal fissures and subsequently in the myotomes, among white muscle lamellae. The cells from the intermyotomal fissure differentiate into fibroblasts. The cells which have migrated into the myotomes, differentiate into mesenchyme-derived myoblasts. After hatching, white muscle lamellae are successively converted into polygonal muscle fibres. Conversion of lamellae into fibres may occur through splitting of muscle lamellae, or cylindrical muscle fibres may arise de novo as a result of fusion of mesenchyme-derived myoblasts. No increase in the number of muscle fibre nuclei is observed either in embryonic or juvenile musculature of N. forsteri. We suggest that until the 53 stage of embryonic development, the increase in muscle mass is accomplished mainly through hyperplasy. Thus, lungfish muscle represents the organizational intermediate between fishes and amphibians. This makes it a useful model to study the evolutionary implications of the mechanisms of muscle development.
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http://dx.doi.org/10.1387/ijdb.072323akDOI Listing
June 2008

Muscle stem cells and model systems for their investigation.

Dev Dyn 2007 Dec;236(12):3332-42

INSERM U384, Clermont-Ferrand, France.

Stem cells are characterized by their clonal ability both to generate differentiated progeny and to undergo self-renewal. Studies of adult mammalian organs have revealed stem cells in practically every tissue. In the adult skeletal muscle, satellite cells are the primary muscle stem cells, responsible for postnatal muscle growth, hypertrophy, and regeneration. In the past decade, several molecular markers have been found that identify satellite cells in quiescent and activated states. However, despite their prime importance, surprisingly little is known about the biology of satellite cells, as their analysis was for a long time hampered by a lack of genetically amenable experimental models where their properties can be dissected. Here, we review how the embryonic origin of satellite cells was discovered using chick and mouse model systems and discuss how cells from other sources can contribute to muscle regeneration. We present evidence for evolutionarily conserved properties of muscle stem cells and their identification in lower vertebrates and in the fruit fly. In Drosophila, muscle stem cells called adult muscle precursors (AMP) can be identified in embryos and in larvae by persistent expression of a myogenic basic helix-loop-helix factor Twist. AMP cells play a crucial role in the Drosophila life cycle, allowing de novo formation and regeneration of adult musculature during metamorphosis. Based on the premise that AMPs represent satellite-like cells of the fruit fly, important insight into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.
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http://dx.doi.org/10.1002/dvdy.21345DOI Listing
December 2007

Shaping leg muscles in Drosophila: role of ladybird, a conserved regulator of appendicular myogenesis.

PLoS One 2006 Dec 27;1:e122. Epub 2006 Dec 27.

Institut National de la Santé et de la Recherche Médicale U384, Faculté de Medecine, Clermont-Ferrand, France.

Legs are locomotor appendages used by a variety of evolutionarily distant vertebrates and invertebrates. The primary biological leg function, locomotion, requires the formation of a specialised appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognised as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultrastructure and functional properties of leg muscles in Drosophila. Ladybird expression is progressively activated in myoblasts associated with the imaginal leg disc and precedes that of the founder cell marker dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties of developing myotubes, impairing their ability to grow and interact with the internal tendons and epithelial attachment sites. It also affects sarcomeric ultrastructure, resulting in reduced leg muscle performance and impaired mobility in surviving flies. The over-expression of ladybird also results in an abnormal pattern of dorsally located leg muscles, indicating different requirements for ladybird in dorsal versus ventral muscles. This differential effect is consistent with the higher level of Ladybird in ventrally located myoblasts and with positive ladybird regulation by extrinsic Wingless signalling from the ventral epithelium. In addition, ladybird expression correlates with that of FGF receptor Heartless and the read-out of FGF signalling downstream of FGF. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fibre formation. A key role for ladybird in leg myogenesis is further supported by its capacity to repress vestigial and to down-regulate the vestigial-governed flight muscle developmental programme. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialised pool of myoblasts expressing ladybird/Lbx1. The ladybird/Lbx1 gene family appears as a part of an ancient genetic circuitry determining leg-specific properties of myoblasts and making an appendage adapted for locomotion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000122PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1762424PMC
December 2006

The origin of syncytial muscle fibres in the myotomes of Xenopus laevis--a revision.

Folia Biol (Krakow) 2005 ;53(1-2):39-44

Department of General Zoology, Zoological Institute, University of Wrocław, Sienkiewicza 21, 53-335 Wrocław, Poland.

During the early stages of myogenesis in X. laevis, the primary myoblasts (of mesodermal origin) differentiate simultaneously, in each myotome, into mononucleate myotubes. At later stages mesenchymal cells appear in intermyotomal fissures and then in the myotomes between myotubes and contribute to the formation ofsyncytial muscle fibres. The pathway of mesenchymals cell during myogenesis was described in X laevis by monitoring the incorporation of 3H-thymidine. 3H-thymidine was incorporated in the nuclei of mesenchymal cells in intermyotomal fissures of younger myotomes and then in those of older myotomes between the myotubes revealing the proliferation of mesenchymal cells. As expected, nuclei of differentiating mononucleate myotubes did not incorporate 3H-thymidine. At later stages of myogenesis the myotubes were found to contain two classes of nuclei: large nuclei of the primary myoblasts (of myotomal origin) and smaller nuclei originating from secondary myoblasts ofmesenchymal origin. TEM and autoradiographic analyses confirm that mulinucleate myotubes in X. laevis arise through fusion of secondary myoblasts with mononucleate myotubes.
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http://dx.doi.org/10.3409/1734916054663401DOI Listing
January 2006

Myotomal myogenesis of axial muscle in the sturgeon Acipenser baeri (Chondrostei, Acipenseriformes).

Folia Biol (Krakow) 2005 ;53(1-2):29-38

Department of General Zoology, Zoological Institute, University of Wrocław, Sienkiewicza 21, 50-335 Wrocław, Poland.

Compared to teleost fishes, a unique character of the myogenesis of the plesiomorphic A. baeri is the fusion of myoblasts derived from the somite, leading to the formation of multinucleate muscle lamellae. Then, the lamellae are converted into cylindrical muscle fibres. The mechanism of transformation of lamellae into fibres is still debatable. Early embryonic muscle growth is mainly due to the hypertrophy of somite-cell derived stock. After hatching, hypertrophic growth occurs parallel to hyperplastic growth. Proliferatively active mesenchymal cells, which migrate from the intermyotomal space into the myotomes, participate in both processes.
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http://dx.doi.org/10.3409/1734916054663537DOI Listing
January 2006

Coordinated development of muscles and tendons of the Drosophila leg.

Development 2004 Dec 10;131(24):6041-51. Epub 2004 Nov 10.

INSERM U.384, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont Ferrand, France.

Since Miller's morphological description, the Drosophila leg musculature and its formation has not been revisited. Here, using a set of GFP markers and confocal microscopy, we analyse Drosophila leg muscle development, and describe all the muscles and tendons present in the adult leg. Importantly, we provide for the first time evidence for tendons located internally within leg segments. By visualising muscle and tendon precursors, we demonstrate that leg muscle development is closely associated with the formation of internal tendons. In the third instars discs, in the vicinity of tendon progenitors, some Twist-positive myoblasts start to express the muscle founder cell marker dumbfounded (duf). Slightly later, in the early pupa, epithelial tendon precursors invaginate inside the developing leg segments, giving rise to the internal string-like tendons. The tendon-associated duf-lacZ-expressing muscle founders are distributed along the invaginating tendon precursors and then fuse with surrounding myoblasts to form syncytial myotubes. At mid-pupation, these myotubes grow towards their epithelial insertion sites, apodemes, and form links between internally located tendons and the leg epithelium. This leads to a stereotyped pattern of multifibre muscles that ensures movement of the adult leg.
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http://dx.doi.org/10.1242/dev.01527DOI Listing
December 2004

Onto-phylogenetic aspect of myotomal myogenesis in Chordata.

Folia Biol (Krakow) 2004 ;52(1-2):1-12

Department of General Zoology, Zoological Institute, University of Wrocław, Sienkiewicza 21, 50-335 Wrocław, Poland.

This paper presents an onto- and phylogenetic aspect of myotoamal myogenesis in Chordata. A comparative analysis of early stages of myotomal myogenesis in Chordata indicates that the myogenic process in this phylum underwent evolutionary changes. The first stage of the process is myogenesis leading to development of mononucleate mature muscle cells, the most advanced stage is formation of multinucleate muscle fibres.
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March 2005

Proliferation and apoptosis in the guinea pig adrenal cortex during postnatal development.

Folia Morphol (Warsz) 2004 Aug;63(3):333-5

Department of Cytophysiology, Histology and Embryology, Medical University, Łódź, Poland.

The aim of the study was to determine the relation between involution of the FZ and the proliferation and apoptosis of the adrenal cortex cells. The study was carried out with adrenal glands from guinea pigs aged 1, 3, 9, 14, 21, 35 and 90 days. Paraffin slices were stained with Mayer haematoxylin and eosin and with acid fuchsin. For the immunohistochemical reaction the Anti -PCNA Clone PC10 (Dako) was used. Apoptosis evaluation was performed with use of the TUNEL method (Roche). The results support the theory of involution of the FZ cells realised mainly by way of apoptosis. Analysis of the distribution and number of PCNA-positive cells in the adrenal cortex on subsequent days of PD may also back up the migration theory of renewal of the adrenal cortex cells.
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August 2004