Down-regulation of neuronal form of Nitric oxide synthase in the Nurse cell of Trichinella spiralis.

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.

The Synthesis of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase (GNE), α-dystroglycan, and ß-galactoside α-2,3-sialyltransferase 6 (ST3Gal6) By Skeletal Muscle Cell As a Response To Infection with Trichinella Spiralis.

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-ß-1,4-Gl-cNAc-ß-1,2-Man-α-1-O-Ser/Thr glycan. The third protein of interest for this study was the enzyme ß-galactoside α-2,3-sialyltransferase 6 (ST3Gal6), which transfers sialic acid preferably onto Gal-ß-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.

Neuroanatomy and behavior in mice with a haploinsufficiency of AT-rich interactive domain 1B (ARID1B) throughout development.

BACKGROUND: One of the causal mechanisms underlying neurodevelopmental disorders (NDDs) is chromatin modification and the genes that regulate chromatin. AT-rich interactive domain 1B (ARID1B), a chromatin modifier, has been linked to autism spectrum disorder and to affect rare and inherited genetic variation in a broad set of NDDs. METHODS: A novel preclinical mouse model of Arid1b deficiency was created and validated to characterize and define neuroanatomical, behavioral and transcriptional phenotypes. Neuroanatomy was assessed ex vivo in adult animals and in vivo longitudinally from birth to adulthood. Behavioral testing was also performed throughout development and tested all aspects of motor, learning, sociability, repetitive behaviors, seizure susceptibility, and general milestones delays. RESULTS: We validated decreased Arid1b mRNA and protein in Arid1b+/- mice, with signatures of increased axonal and synaptic gene expression, decreased transcriptional regulator and RNA processing expression in adult Arid1b+/- cerebellum. During neonatal development, Arid1b+/- mice exhibited robust impairments in ultrasonic vocalizations (USVs) and metrics of developmental growth. In addition, a striking sex effect was observed neuroanatomically throughout development. Behaviorally, as adults, Arid1b+/- mice showed low motor skills in open field exploration and normal three-chambered approach. Arid1b+/- mice had learning and memory deficits in novel object recognition but not in visual discrimination and reversal touchscreen tasks. Social interactions in the male-female social dyad with USVs revealed social deficits on some but not all parameters. No repetitive behaviors were observed. Brains of adult Arid1b+/- mice had a smaller cerebellum and a larger hippocampus and corpus callosum. The corpus callosum increase seen here contrasts previous reports which highlight losses in corpus callosum volume in mice and humans. LIMITATIONS: The behavior and neuroimaging analyses were done on separate cohorts of mice, which did not allow a direct correlation between the imaging and behavioral findings, and the transcriptomic analysis was exploratory, with no validation of altered expression beyond Arid1b. CONCLUSIONS: This study represents a full validation and investigation of a novel model of Arid1b+/- haploinsufficiency throughout development and highlights the importance of examining both sexes throughout development in NDDs.

Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS.

[Three years experience of using mycosyst]. / Nashiiat trigodishen opit ot prilozhenieto na preparata mycosyst.

The aim of this study was to estimate therapeutical potentialities of the Mycosyst (Fluconazole) preparation, of the Gedeon-Richter company (Budapest, Hungary), in women with data for mycotic colpitis. 157 patients for three years period (April 2001-2004) were included in this study. Two groups of patients were formed. In 104 patients mycotic colpitis was diagnosed during curettage for feticide or missed abortion. Another 53 women were treated for mycotic colpitis unrelated to pregnancy. Complains of the patients disappear by the 48-th hour and in almost all women there were not complaints by the 4-th day. The most quickly were influenced itching, discomfort, erythema and dyspareunia and slowly and hardly discharge had been influenced. Mycosyst is a broad spectrum antimycotic agent of bistriazol type. It has good absorption and tissue saturation. 1-2 hours after oral administration maximal plasma concentration is achieved. Plasma half life is about 30 hours. This gave us opportunity for single or two doses Mycosyst for vaginal candidosis treatment. Use of two doses of 150 mg. was enough for acute forms treatment in almost 95% of cases. Mycosyst is sure, easy for use broad spectrum antimycotic from the azole group. This preparation is comparatively cheep and in the average Bulgarian income possibilities.

[Utrogestan and high risk pregnancy]. / Utrogestan i zastrashena bremennost.

Utrogestan is a modern progesterone, which shows maximal effectiveness with minimal side effects. It is a natural progesterone in micronized form, which makes it suitable for oral administration and vaginal application with same effectiveness. The aim of our retrospective study was to evaluate the therapeutical effects of Utrogestan in women with threatened abortion in the first trimester. Our experience dated from about one year and a half. Sixty eight women were treated for threatened abortion with a daily dose of 400 mg Utrogestan. The treatment continued at least 14 days/average 21 days/. Utrogestan was administered orally twice daily. The main indications were first or consecutive threatened spontaneous abortion in first trimester. For the period of time no side effects and subjective complaints were established except particular cases of slight headache and dizziness after morning application. Sixty one of the sixty eight women were dehospitalised with healthy pregnancy with no side effects. In our experience we preferred to use the drug in women with lutein insufficiency before pregnancy. We conclude that Utrogestan can be widely applied in Obstetrics with the proper indications, such as threatened abortion in the first trimester. The prophylactic treatment in women with slighter complaints like dull pain and weight Utrogestan is applied one tablet twice daily. In graver cases the starting dosage is two tablets two or three times daily.

Fas-FasL interaction modulates nitric oxide production in Trypanosoma cruzi-infected mice.

During acute Trypanosoma cruzi infection in mice, many leucocytes undergo apoptosis. Although apoptosis has been ascribed to increased levels of nitric oxide (NO) and Fas-FasL interaction, the importance of this phenomenon in modulating the host response against T. cruzi is unknown. Herein, the role of NO- and Fas-FasL-induced apoptosis in modulating the immune response to T. cruzi was evaluated using mice deficient in Fas expression (MRL/MpJ-Fas lpr) and inducible nitric oxide synthase (iNOS) knockout mice (iNOS-/-). The results showed that besides decreasing apoptosis induction after infection, impairment of the Fas-FasL interaction resulted in decreased NO production, as a consequence of enhanced T helper 2 (Th2) cytokine production. Differently, blockage of NO-induced apoptosis resulted in uncontrolled cytokine production, rather than a biased Th2 cytokine pattern. Together, these results suggested that Fas and FasL-induced apoptosis could be implied in modulation of the immune response against T. cruzi by interfering with cytokine and NO production during the acute phase of the infection.

The role of endothelin in the pathogenesis of Chagas' disease.

Infection with Trypanosoma cruzi causes a generalised vasculitis of several vascular beds. This vasculopathy is manifested by vasospasm, reduced blood flow, focal ischaemia, platelet thrombi, increased platelet aggregation and elevated plasma levels of thromboxane A(2) and endothelin-1. In the myocardium of infected mice, myonecrosis and a vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium are observed. Immunohistochemistry studies employing anti-endothelin-1 antibody revealed increased expression of endothelin-1, most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for prepro endothelin-1, endothelin converting enzyme and endothelin-1 were observed in the infected myocardium. When T. cruzi-infected mice were treated with phosphoramidon, an inhibitor of endothelin converting enzyme, there was a decrease in heart size and severity of pathology. Mitogen-activated protein kinases and the transcription factor activator-protein-1 regulate the expression of endothelin-1. Therefore, we examined the activation of mitogen-activated protein kinases in the myocardium by T. cruzi. Western blot demonstrated an extracellular signal regulated kinase. In addition, the activator-protein-1 DNA binding activity, as determined by electrophoretic mobility shift assay, was increased. Increased expression of cyclins A and cyclin D1 was observed in the myocardium, and immunohistochemistry studies revealed that interstitial cells and vascular and endocardial endothelial cells stained intensely with antibodies to these cyclins. These data demonstrate that T. cruzi infection of the myocardium activates extracellular signal regulated kinase, activator-protein-1, endothelin-1, and cyclins. The activation of these pathways is likely to contribute to the pathogenesis of chagasic heart disease. These experimental observations suggest that the vasculature plays a role in the pathogenesis of chagasic cardiomyopathy. Additionally, the identification of these pathways provides possible targets for therapeutic interventions to ameliorate or prevent the development of cardiomyopathy during T. cruzi infection.

Trypanosoma cruzi infection (Chagas' disease) of mice causes activation of the mitogen-activated protein kinase cascade and expression of endothelin-1 in the myocardium.

Chagas' disease, caused by the parasite Trypanosoma cruzi, is an important cause of heart disease. Previous studies from this laboratory revealed that microvascular spasm and myocardial ischemia were observed in infected mice. Infection of endothelial cells with this parasite increased the synthesis of biologically active endothelin-1 (ET-1). Therefore. in the myocardium of T. cruzi-infected mice, we examined ET-1 expression and the p42/44-mitogen activated protein kinase (MAPK)-AP-1 pathway that regulates the expression of ET-1. There was parasitism and myonecrosis in the myocardium of infected C57BL/6 mice. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed elevated mRNA expression of transcription factor AP-1 (c-jun and c-fos) and increased AP-1 DNA binding activity as determined by electrophoretic mobility shift assay (EMSA). Western blot analysis demonstrated an increase in the phosphorylated forms of extracellular signal-regulated kinase (ERK1/2). ET-1 mRNA was upregulated in the myocardium of infected mice. Immunohistochemical and immunoelectron microscopy using anti-ET-1 antibody detected increased expression in cardiac myocytes and endothelium of these mice. These data suggest that ET-1 contributes to chagasic cardiomyopathy and that the mechanism of the increased expression of ET-1 is a result of the activation of the MAPK pathway by T. cruzi infection.

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.

Cell cycle molecules and diseases of the cardiovascular system.

Injury to the cardiovascular system causes an elevated expression of endothelin-1 (ET-1) and activation of several important signaling pathways including the mitogen-activated kinase (MAPK) cascade. The activation of these pathways has been implicated in the pathogenesis of cardiovascular disease caused by hypoxia, infections, and ischemia /reperfusion injury, cardiomyopathy and restenosis after balloon angioplasty. Important downstream targets of the MAPK and ET-1 pathways are the cell cycle regulatory molecules (cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors). Regulation of these molecules contributes to remodeling throughout the cardiovascular system. In addition, cell cycle molecules are important in the regulation of angiogenesis. These new data have led to the development of potential therapeutic modalities targeting these regulatory molecules in order to ameliorate various cardiovascular disease states.

The induction of DNA strand breaks at specific sites by N-nitroso-N-ethylurea depends on the phases of the cell cycle.

Synchronized root meristems of Pisum sativum were treated at each phase of the cell cycle with 6.25 mM N-nitroso-N-ethylurea. DNA extracted from treated cells and run in agarose gel electrophoresis exhibits a series of discrete fragments with length below 2500 bp and a significant number of unspecific single-stranded breaks (or alkali-labile sites). Experiments with micrococcal nuclease indicated that the nucleosomal organization of the chromatin is not responsible for the generation of the discrete fragments: it seems that their appearance is associated with a preferable attack of the mutagen at specific sites, characteristic for the plant genome. Moreover, a cell cycle dependent release of the discrete fragments was found with maximum at G1-S and minimum at mitosis. The model experiments designed to clarify this observation suggest that it might be determined from the cell cycle dependent fluctuation in the accessibility of the chromatin DNA and/or the process of excision-repair.

Immunochemical prognosis of heterosis in Zea mays.

Heterosis is a complex biological phenomenon. Because of the complex interaction and interrelation between "genes - metabolism - environment", it is hardly possible to expect a clarification of the heterosis phenomenon through simple genetic explanations only (Hagemann et al. 1967).We have followed an immunochemical aspect and method of research. The antigenic analysis of inbred lines and their hybrids was used for studying heterosis and for investigating the possibilities for its prognosis.The heterosis effect was proved under field conditions. Our investigations (Dimitrov et al. 1972) show the presence of four protein fractions in the seed extracts of inbred maize lines, while the heterotic hybrids contain a fifth protein fraction. Antigenie analysis by the method of Grabar and Williams was carried out for a more complete characterization and determination of the specificity of the fractions obtained (Grabar and Williams 1955).The present publication is a result of our research upon maize inbred lines, simple heterotic and nonheterotic hybrids and their backcrosses. A double diffusion in agar gel, according to the Ouchterlony method (Ouchterlony 1958), confirmed the presence of a fifth protein fraction in the heterotic hybrids, which can not be found in the inbred lines or in the nonheterotic hybrids.In the inbred lines we found 3 protein fractions common to all of them, and also a fourth (individual antigen), contained only in the inbred lines that produce a heterosis effect when crossed. It was determined that the carrier of the information for the synthesis of the individual antigen is a nuclear factor.We also determined the conditions under which (after direct and inverse crosses and after crosses in one direction only) heterotic hybrids are obtained. Some backcrosses show a marked heterosis effect connected with the doubling of the factor, carrier of the information for the individual antigen. This fact is important for the scientific verification of the methods for obtaining complex heterotic hybrids.Our results throw some light upon the genetic nature of the heterosis phenomenon.The heterosis effect was determined only for inbred lines whose seed extracts have a precipitation arc against their homologous serum, absorbed with the extract of its partner. This allows for the prognosis of heterosis in maize, i.e. for the determination in advance (through the double immunodiffusion) of the inbred lines and also of the direction of crosses that produce the heterosis effect.The present publication is a continuation of our report (Dimitrov et al. 1972) concerning the research on the nature of heterosis in connection with its prognosis. In our investigation we have accepted as heterotic only hybrids whose seed yield exceeds the seed yields of each of the separate inbred lines being crossed.

Immunoelectrophoretic studies of heterosis effect in Zea mays.

The present report is based on tests using antigenic analysis of inbred lines and their hybrids for the prognosis of heterosis (hybrid vigour) in Zea mays.Inbred lines, whose hybrid vigour has been proved under field conditions, and their hybrids were characterized by the method of Grabar and Williams.The results of the electrophoretic and immunoelectrophoretic investigations show that the extracts from maize seeds of the inbred lines C-103, N-6 and WF-9 contain four protein fractions, while those of the hybrids N-6 x C-103 and WF-9 x N-6 also contain a fifth fraction.The immunoelectrophoretic pattern of the extracts from the hybrid seeds N-6 x C-103 shows that, with their homologous serum, they give a precipitation line (arc X) which is not obtained by the interaction of the parent line extracts with homologous and heterologous serums.In this case we probably have a protein synthesis in the hybrid which is caused by the deblocking of some links in the biosynthetic apparatus of the inbred lines.It was established that the extracts from the hybrid N-6 x C-103 gave a larger number of precipitation arcs with the heterologous serum anti C-103.The characterization of the antigenic structure of the inbred lines enables one to determine not only the fractions common to them but also the number of those fractions by which they differ from one another. Proceeding in this manner, one could accomplish an immunoelectrophoretic prognosis of heterosis and penetrate deeper into its essence.