Rositsa Milcheva

Summary: The free radical nitric oxide (NO) and Ca²⁺ are critical regulators of skeletal muscle exercise performance and fatigue. The major source of NO in skeletal muscle cells is the neuronal form of the enzyme Nitric oxide synthase (nNOS). One of the most peculiar characteristics of the Nurse cell of Trichinella spiralis (T. spiralis) is the complete loss of the contractile capabilities of its derivative striated muscle fiber. The aim of the present study was to clarify the expression of nNOS protein and mRNA in striated muscles during the muscle phase of T. spiralis infection in mice. Muscle tissue samples were collected from mice at days 0, 14, 24, and 35 post infection (d.p.i.). The expression of nNOS was investigated by immunohistochemistry, and the expression levels of mRNA of mouse Nitric oxide synthase 1 (Nos1) by real-time PCR. The presence of nNOS protein was still well observable in the disintegrated sarcoplasm at the early stage of infection. The cytoplasm of the developing and mature Nurse cell showed the absence of this protein. At least at the beginning of the Nurse cell development, Trichinella uses the same repairing process of skeletal muscle cell, induced after any trauma and this corroborates very well our results concerning the nNOS expression on day 14 p.i. At a later stage, however, we could suggest that the down-regulation of nNOS in the Nurse cell of T. spiralis either serves a protective function or is an outcome of the genetic identity of the Nurse cell.

Purpose Dystrophin and the dystrophin glycoprotein complex serve as a cytoskeletal integrator, critical for muscle membrane stability. The aim of the present study was to clarify the expression of dystrophin protein and mRNA in the skeletal muscle tissue during the muscle phase of trichinellosis in mice. Methods Muscle tissue was collected from mice experimentally infected with Trichinella spiralis at days 0, 14 and 40 after infection. The expression of dystrophin in the muscle tissue was investigated by immunohistochemistry with antibodies against three different domains of the protein, and the expression levels of Dys mRNA by real-time PCR. Results The presence of dystrophin protein was increased in the de-differentiating cytoplasm at the early stage of muscle infection and was persisting also in the mature Nurse cell harbouring the parasite. It was accompanied by significantly elevated expression of Dys mRNA at days 14 and 40 after infection. Conclusion Our findings indicate that dystrophin plays a role in regeneration of the muscle and in the Nurse cell formation and stability for security of the parasite survival.

The study reports the synthesis and characterization of novel triple stimuli responsive interpenetrating polymer network (IPN) based on two polyzwitterionic networks, namely of poly(carboxybetaine methacrylate) and poly(sulfobetaine methacrylate). The zwitterionic IPN hydrogel demonstrates the ability to expand or shrink in response to changes in three “biological” external stimuli such as temperature, pH, and salt concentration. The IPN hydrogel shows good mechanical stability. In addition, other important features such as non-cytotoxicity and antibiofouling activity against three widespread bacteria as P. Aeruginosa, A. Baumanii, and K. Pneumoniae are demonstrated. The in vivo behavior of the novel zwitterionic IPN hydrogel suggests that this smart material has very good potential as a biomaterial.

Summary: The Nurse cell of the parasitic nematode Trichinella spiralis is a unique structure established after genetic, morphological and functional modification of a small portion of invaded skeletal muscle fiber. Even if the newly developed cytoplasm of the Nurse cell is no longer contractile, this structure remains well integrated within the surrounding healthy tissue. Our previous reports suggested that this process is accompanied by an increased local biosynthesis of sialylated glycoproteins. In this work we examined the expressions of three proteins, functionally associated with the process of sialylation. The enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key initiator of the sialic acid biosynthetic pathway. The α-dystroglycan was the only identified sialylated glycoprotein in skeletal muscles by now, bearing sialyl-α-2,3-Gal-β-1,4-Gl-cNAc-β-1,2-Man-α-1-O-Ser/Thr glycan. The third protein of interest for this study was the enzyme β-galactoside α-2,3-sialyltransferase 6 (ST3Gal6), which transfers sialic acid preferably onto Gal-β-1,4-GlcNAc as an acceptor, and thus it was considered as a suitable candidate for the sialylation of the α-dystroglycan. The expressions of the three proteins were analyzed by real time-PCR and immunohistochemistry on modified methacarn fixed paraffin tissue sections of mouse skeletal muscle samples collected at days 0, 14 and 35 post infection. According to our findings, the up-regulation of GNE was a characteristic of the early and the late stage of the Nurse cell development. Additional features of this process were the elevated expressions of α-dystroglycan and the enzyme ST3Gal6. We provided strong evidence that an increased local synthesis of sialic acids is a trait of the Nurse cell of T. spiralis, and at least in part due to an overexpression of α-dystroglycan. In addition, circumstantially we suggest that the enzyme ST3Gal6 is engaged in the process of sialylation of the major oligosaccharide component of α-dystroglycan.

In skeletal muscles, the sialic acids have a great significance for their functional maintenance and proper structural organization. Our work described the expressions of St3gal, St6gal and St6galnac sialyltransferases specific for glycoproteins in mouse skeletal muscles and murine C2C12 myotubes. Lectin histochemistry, cytochemistry and lectin blot were used to demonstrate the membrane localization and the electrophoretic profiles of α-2,3- and α-2,6-sialylated glycoproteins. The expression levels of sialyltransferases were analysed by real time RT-PCR and western blot. The enzymes St6gal2 and St6galnac1 were not expressed in skeletal muscle tissue and C2C12 myotubes. In both experimental groups, mRNAs of the St3gal family prevailed over the mRNA expressions of the St6gal and St6galnac families. The profiles of sialyltransferase expressions showed differences between the two experimental groups, illustrated by the absence of expressions of the mRNA for the St3gal6 and St6galnac3 genes in the C2C12 cell samples and by the different shares of the enzymes St3gal3 and St3gal4 in both experimental groups. The different patterns of enzyme expressions in both experimental groups corresponded with differences between their α-2,3- and α-2,6-sialylated glycoprotein profiles. These results could be a useful addendum to the knowledge concerning the glycosylation of the skeletal muscle tissue.

In skeletal muscles, the sialic acids have a great significance for their functional maintenance and proper structural organization. Our work described the expressions of St3gal, St6gal and St6galnac sialyltransferases specific for glycoproteins in mouse skeletal muscles and murine C2C12 myotubes. Lectin histochemistry, cytochemistry and lectin blot were used to demonstrate the membrane localization and the electrophoretic profiles of α-2,3- and α-2,6-sialylated glycoproteins. The expression levels of sialyltransferases were analysed by real time RT-PCR and western blot. The enzymes St6gal2 and St6galnac1 were not expressed in skeletal muscle tissue and C2C12 myotubes. In both experimental groups, mRNAs of the St3gal family prevailed over the mRNA expressions of the St6gal and St6galnac families. The profiles of sialyltransferase expressions showed differences between the two experimental groups, illustrated by the absence of expressions of the mRNA for the St3gal6 and St6galnac3 genes in the C2C12 cell samples and by the different shares of the enzymes St3gal3 and St3gal4 in both experimental groups. The different patterns of enzyme expressions in both experimental groups corresponded with differences between their α-2,3- and α-2,6-sialylated glycoprotein profiles. These results could be a useful addendum to the knowledge concerning the glycosylation of the skeletal muscle tissue.

I. Background. In skeletal muscles the sialic acids have a great significance for their functional maintenance and proper structural organization. Our work for the first time described the expressions of ST3Gal, ST6Gal and ST6GalNAc sialyltransferases specific for glycoproteins in mouse skeletal muscles and murine C2C12 myotube cell cultures.II. Methods and Results. Lectin histochemistry, cytochemistry and lectin blot were used to demonstrate the membrane localization and the electrophoretic profiles of α-2,3- and α-2,6-sialylated glycoproteins. The expression levels of sialyltransferases were analyzed by real time RT-PCR and western blot. The enzymes ST6Gal2 and ST6GalNAc1 were not expressed in skeletal muscle tissue and C2C12 myotubes. In both experimental groups mRNAs of the ST3Gal family prevailed over the mRNA expressions of the ST6Gal and ST6GalNAc families. The profiles of STR expressions showed differences between the two experimental groups, illustrated by the absence of expressions of the mRNA for the ST3Gal6 and ST3GalNAc3 enzymes in the C2C12 cell samples and by the different shares of the enzymes ST3Gal3 and ST3Gal4 in both experimental groups. The different patterns of enzyme expressions in both experimental groups corresponded with differences between their α-2,3- and α-2,6-sialylated glycoprotein profiles.III. Conclusions. These results could be a useful addendum to the knowledge concerning the glycosylation of the skeletal muscle tissue. In addition, this report would be helpful and informative for any researches in future where the C2C12 myotube cell cultures will take a place as an experimental model.

The sialylation of the glycoproteins in skeletal muscle tissue is not well investigated, even though the essential role of the sialic acids for the proper muscular function has been proven by many researchers. The invasion of the parasitic nematode Trichinella spiralis in the muscles with subsequent formation of Nurse cell-parasite complex initiates increased accumulation of sialylated glycoproteins within the affected area of the muscle fiber. The aim of this study is to describe some details of the α-2,6-sialylation in invaded muscle cells. Asynchronous invasion with infectious T. spiralis larvae was experimentally induced in mice. The areas of the occupied sarcoplasm were reactive towards α-2,6-sialic acid specific Sambucus nigra agglutinin during the whole process of transformation to a Nurse cell.The cytoplasm of the developing Nurse cell reacted with Helix pomatia agglutinin, Arachis hypogea agglutinin and Vicia villosa lectin-B4 after neuraminidase pretreatment.Up-regulation of the enzyme ST6GalNAc1 and down-regulation of the enzyme ST6GalNAc3 were detected throughout the course of this study. The results from our study assumed accumulation of sialyl-Tn-Ag, 6`-sialyl lactosamine, SiA-α-2,6-Gal-β-1,3-GalNAc-α-Ser/Thr and Gal-β-1,3-GalNAc(SiA-α-2,6-)-α-1-Ser/Thr oligosaccharide structures into the occupied sarcoplasm. Further investigations in this domain will develop the understanding about the amazing adaptive capabilities of skeletal muscle tissue.

The aim of the work was to analyze oligosaccharide composition with the focus on O-linked glycoproteins presence in the total extract obtained from different Trichinella species muscle larvae by means of lectin affino-blot with lectins selected for their sugar specificity. The absence of reactivity with two lectins, Tritrichomonas mobilensis lectin and Maackia amurensis agglutinin, indicated that the species of the Trichinella genus do not synthesize sialic acid. Reactivity with Helix pomatia lectin, Vicia villosa lectin-B4, peanut agglutinin and Ulex europeus lectin-I identified the presence of O-linked glycans identical to carcinoma-associated Tn-antigen (GalNAc-α-Ser/Thr) and T-antigen (Gal-β1,3-GalNAc-α-Ser/Thr) and also structures analogous to ABH-blood group antigens. The results obtained may contribute to a better understanding of the glycobiology of this parasitic nematode in relation to occupation of its intracellular niches.