Glycosylation changes in different developmental stages of Trichinella

The in situ identification of carbohydrate structures in Trichinella spiralis intestinal larvae, adults and L1 muscular larvae was carried out by lectin histochemistry, with emphasis on the O-linked glycans. The absence of reactivity with two lectins-TML and MAL indicated that Trichinella spiralis does not synthesize sialic acid. Reactivity with HPA, VVL-B4, PNA and UEA-I staining suggested that T. spiralis synthesizes and expresses on its cuticle O-linked glycans analogous to Tn-antigen (GalNAc-α-Ser/Thr), T-antigen (Gal-β1,3-GalNAc-α-Ser/Thr) and also structures analogous to A-blood group antigens (GalNAc-α1,3-Gal-β1,3(4)-(Fuc-α1,2-)-R). Expression of the saccharidic moieties is stage-specific. Blood group-A and T-antigen structures were identified on the cuticle of the intestinal and muscular larvae. The Tn-antigen structure was missing in the intestinal larvae. Appropriate ligands for WGA were not identified in the adult individuals. The obtained results may contribute to a better understanding of the glycobiology of this parasitic nematode in relation to occupation of its intracellular niche. The presence of saccharidic structures analogous to some of those expressed on the intestinal epithelial cells may serve as a protective shield on the surface of the parasite.     

Antioxidant activity of fluoxetine: Studies in mice melanoma model

In vivo effects of the antidepressant fluoxetine on spleen antioxidant status of C57BL/6 mice were studied using a melanoma experimental model. After a 14‐day treatment with fluoxetine (10 mg kg^?1 day^?1, i.p.), the endogenous antioxidant non‐enzyme (glutathione) and enzyme (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) defense systems in spleen of healthy animals were not changed; the lipid peroxidation (LP) was also unchanged. When B16F10 melanoma cells were introduced in C57BL/6 mice 2 h before fluoxetine treatment, a drug‐protective effect against the melanoma‐induced oxidative changes (increased LP and decreased total glutathione (GSH)‐level, as well as antioxidant enzyme activities) in spleen was observed. Fluoxetine dose‐dependently reduced the amounts of free oxygen radicals (hydroxyl and superoxide anion radicals), generated in chemical systems. Taken together, the present results suggest that fluoxetine, acting as antioxidant, prevents from melanoma‐induced oxidative changes in mice spleen. Copyright © 2010 John Wiley & Sons, Ltd.     

Glycation and post-translational processing of human interferon-gamma expressed in Escherichia coli

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-gamma during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140/Arg141, Phe137/Arg138, Met135/Leu136, and Lys131/Arg132) and two minor (Lys109/Ala110 and Arg90/Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-gamma originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-gamma, whereas covalent dimerization completely abolished it.     

Hierarchical genetic algorithm versus static optimization-investigation of elbow flexion and extension movements

The applicability of static optimization (and, respectively, frequently used objective functions) for prediction of individual muscle forces for dynamic conditions has often been discussed. Some of the problems are whether time-independent objective functions are suitable, and how to incorporate muscle physiology in models. The present paper deals with a twofold task: (1) implementation of hierarchical genetic algorithm (HGA) based on the properties of the motor units (MUs) twitches, and using multi-objective, time-dependent optimization functions; and (2) comparison of the results of the HGA application with those obtained through static optimization with a criterion "minimum of a weighted sum of the muscle forces raised to the power of n". HGA and its software implementation are presented. The moments of neural stimulation of all MUs are design variables coding the problem in the terms of HGA. The main idea is in using genetic operations to find these moments, so that the sum of MUs twitches satisfies the imposed goals (required joint moments, minimal sum of muscle forces, etc.). Elbow flexion and extension movements with different velocities are considered as proper illustration. It is supposed that they are performed by two extensor muscles and three flexor muscles. The results show that HGA is a suitable means for precise investigation of motor control. Many experimentally observed phenomena (such as antagonistic co-contraction, three-phasic behavior of the muscles during fast movements) can find their explanation by the properties of the MUs twitches. Static optimization is also able to predict three-phasic behavior and could be used as practicable and computationally inexpensive method for total estimation of the muscle forces.     

Atypical behaviour and survival of Streptococcus pyogenes L forms during intraperitoneal infection in rats

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.     

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

Naringenin (NGEN), a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth. It has been also shown to access the brain and there is an increasing interest in its therapeutic applications. The up-regulated expression of Cx43 leads to the suppression of tumorigenicity with promoted apoptotic events. In this study, we investigated the in vivo effect of NGEN in fostering apoptosis in cerebrally implanted C6 glioma cells rat model. We analysed the expression of Cx43, caspase-3, caspase-9, Cyt C, Bcl-2 and Bax in vivo by immunoblot analysis and the ultra structure of brain cells by transmission electron microscopy. Supplementation of NGEN to experimental animals modulated Bcl-2/Bax ratio and up-regulation of caspase-3 and 9. NGEN was also found to up-regulate the expression of Cx43. These findings provide evidence that NGEN’s apoptotic effect, modulation of Bcl-2/Bax ratio leads to release of Cyt C from mitochondria, thereby activation of caspase-3 and caspase-9 is mediated by enhanced expression of Cx43. These observations were well supported by the transmission electron microscopic results which showed the characteristic apoptotic features. In conclusion, our findings demonstrate that NGEN promotes apoptosis in rat C6 glioma model.     

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli

Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.     

Experimental and modelling investigation of learning a fast elbow flexion in the horizontal plane

Changes in the kinematic and electromyographic characteristics that occur while learning to move as fast as possible have been studied experimentally. Experimental investigation of what happens to the individual motor units (MUs) is more difficult. Access to each MU is impossible, and the recruitment and force developing properties of all individual MUs cannot be known. Thus, what is currently known about MU firing is based on experiments that have recorded relatively few MUs compared to what exists in the entire muscle. A recently developed muscle model (Raikova and Aladjov, 2002, J. Biomechanics, 35, 1123-1135) composed of MUs with different properties can be used for such investigation. The process of learning fast elbow flexion in the horizontal plane was simulated and the results were compared with experimentally measured data. Comparing the simulation results of the very first trial of a particular subject with those of the last trail (at the end of the learning process), it can be concluded that the speed of limb motion and muscle forces increase initially as a result of the more synchronous MUs activation and the increase of firing rate of active MUs. Further improvement necessitated an appreciable reduction in the motor task requirements (i.e. less muscle force and less MUs' activity) set in the computational algorithm by optimization criteria. This forced the next process-inclusion of additional MUs.     

<Emphasis Type="Italic">Drosophila</Emphasis> as a platform to predict the pathogenicity of novel aminoacyl-tRNA synthetase mutations in CMT

Charcot-Marie-Tooth disease (CMT) is the major form of inherited peripheral neuropathy in humans. CMT is clinically and genetically heterogeneous and four aminoacyl-tRNA synthetases have been implicated in disease etiology. Mutations in the YARS gene encoding a tyrosyl-tRNA synthetase (TyrRS) lead to Dominant Intermediate CMT type C (DI-CMTC). Three dominant YARS mutations were so far associated with DI-CMTC. To further expand the spectrum of CMT causing genetic defects in this tRNA synthetase, we performed DNA sequencing of YARS coding regions in a cohort of 181 patients with various types of peripheral neuropathy. We identified a novel K265N substitution that in contrast to all previously described mutations is located at the anticodon recognition domain of the enzyme. Further genetic analysis revealed that this variant represents a benign substitution. Using our recently developed DI-CMTC Drosophila model, we tested in vivo the pathogenicity of this new YARS variant. We demonstrated that the developmental and behavioral defects induced by all DI-CMTC causing mutations were not present upon ubiquitous or panneuronal TyrRS K265N expression. Thus, in line with our genetic studies, functional analysis confirmed that the K265N substitution does not induce toxicity signs in Drosophila. The consistency observed throughout this work underscores the robustness of our DI-CMTC animal model and identifies Drosophila as a valid read-out platform to ascertain the pathogenicity of novel mutations to be identified in the future.     

Investigating micronucleus assay applicability for prediction of normal tissue intrinsic radiosensitivity in gynecological cancer patients

BackgroundPelvic organs morbidity after irradiation of cancer patients remains a major problem although new technologies have been developed and implemented. A relatively simple and suitable method for routine clinical practice is needed for preliminary assessment of normal tissue intrinsic radiosensitivity. The micronucleus test (MNT) determines the frequency of the radiation induced micronuclei (MN) in peripheral blood lymphocytes, which could serve as an indicator of intrinsic cell radiosensitivity.AimTo investigate a possible use of the micronucleus test (MNT) for acute radiation morbidity prediction in gynecological cancer patients.Materials and methodsForty gynecological cancer patients received 50 Gy conventional external pelvic irradiation after radical surgery. A four-field “box” technique was applied with 2D planning. The control group included 10 healthy females.Acute normal tissue reactions were graded according to NCI CTCAE v.3.0. From all reaction scores, the highest score named “summarized clinical radiosensitivity” was selected for a statistical analysis.MNT was performed before and after in vitro irradiation with 1.5 Gy. The mean radiation induced frequency of micronuclei per 1000 binucleated cells (MN/1000) and lymphocytes containing micronuclei per 1000 binucleated cells (cells with MN/1000) were evaluated for both patients and controls.An arbitrary cut off value was created to pick up a radiosensitive individual: the mean value of spontaneous frequency of cells with MN/1000 ± 2SD, found in the control group.ResultsBoth mean spontaneous frequency of cells with MN/1000 and MN/1000 were registered to be significantly higher in cancer patients compared to the control group (t = 2.46, p = 0.02 and t = 2.51, p = 0.02). No statistical difference was registered when comparing radiation induced MN frequencies between those groups.Eighty percent (32) of patients developed grade 2 summarized clinical radiosensitivity, with great variations in MNT parameters. Only three patients with grade 2 “summarized clinical radiosensitivity” had values of cells with MN/1000 above the chosen radiosensitivity threshold.ConclusionThe present study was not able to confirm in vitro MNT applicability for radiosensitivity prediction in pelvic irradiation.