Draft genome sequences data of two Rosemountvirus phages isolated from soil near poultry farm.

Here we report the draft complete genome sequence of Escherichia phage vB_EcoM_IntR and Escherichia phage vB_EcoM_PiR isolated from a poultry farm soil sample. Bacteriophage isolation on the model of the pathogenic for birds Escherichia coli was carried out. Sequencing was carried out on the Illumina platform. Phage genomes were assembled using a software Geneious package. Phage sequences do not contain tRNA. Phages are a linear double-stranded DNA from Rosemountvirus genus of Myoviridae family with a genome 52,782 and 52,936 bp, respectively, containing 71 predicted open reading frames (ORFs). The phages have a standard icosahedral head and a contractive tail characteristic of the myovirus family.

Adjuvant activity of multimolecular complexes based on Glycyrrhiza glabra saponins, lipids, and influenza virus glycoproteins.

Numerous studies have shown that immunostimulatory complexes containing Quil-A saponin and various antigens are effective in stimulating the immune response and can be used as vaccine preparations for animals and humans. However, Quil-A saponin possesses toxicity and haemolytic activity. In the present work, a saponin-containing preparation named "Glabilox" was isolated from the roots of a Glycyrrhiza glabra L. plant by high-performance liquid chromatography (HPLC). The results showed that Glabilox has no toxicity or haemolytic activity and can form stable immunostimulatory complexes. Subcutaneous immunization of mice with an immunostimulating complex containing Glabilox and H7N1 influenza virus antigens stimulated high levels of humoral and cellular immunity. Vaccination of chickens with the same immunostimulating complex protected 100% of the animals after experimental infection with a homologous virus. Comparative studies showed that the immunogenic and protective activity of immunostimulatory complexes containing Quil-A and immunostimulatory complexes containing Glabilox are comparable to each other. The results of these studies indicated that Glycyrrhiza glabra saponins show great promise as safe and effective adjuvants.

Adjuvant activity of saponins from Kazakhstani plants on the immune responses to subunit influenza vaccine.

In recent years, there have been a number of reports on the successful use of immunostimulatory complexes with saponins and viral glycoproteins as veterinary vaccines and in clinical trials for human medicine. The saponins Algiox, Sapanox and Pangisan were isolated and purified by HPLC from Allochrusa gypsophiloides, Saponaria officinalis and Gypsophila paniculata plants in Kazakhstan and they proved to have low toxicity in experiments with mice, chickens and chicken embryos. Algiox, Sapanox and Pangisan can be used to create immunostimulatory complexes (ISCOMs) similar to saponin-Quil-A-containing ISCOMs both in structure and in immunostimulatory efficiency. The adjuvant effect of the obtained saponins was studied by subcutaneous injection of mice with ISCOMs containing these herbal saponins and lipids and glycoproteins of H7N1 influenza virus. Sapanox, Pangisan and Algiox from Kazakhstani plants of the family Caryophyllaceae could be considered an additional source of highly effective adjuvants not only for veterinary vaccines but also for human medicine.

Biglycan is a novel binding partner of fibroblast growth factor receptor 3c (FGFR3c) in the human testis.

Regulation of spermatogonial maintenance in the human testis is currently not well understood. One pathway suggested to be involved is activated by fibroblast growth factor receptor 3 (FGFR3), which is expressed in a subset of spermatogonia. FGFR3-activating mutations have been identified in spermatocytic seminoma, thought to originate from clonal expansion of spermatogonia. In this study we aimed to characterize potential binding partners of FGFR3, and specifically its mesenchymal "c" splice isoform, in human spermatogonia. Based on expression patterns and homology to the binding site, we identified FGF1, FGF2, and FGF9 as the best candidates for natural ligands of FGFR3c in the testis. In addition, we screened non-FGF proteins and found that a proteoglycan biglycan (BGN) contains a sequence homologous to the FGFR3c binding site on FGF1, and is expressed in peritubular cells adjacent to FGFR3-expressing spermatogonia. Experiments in a cell-free system confirmed that BGN binds to FGFR3c and FGF1. In conclusion, our findings further clarify the complex regulation of FGFR3c in the human testis. We postulate that BGN is a factor secreted by peritubular cells to modulate FGFR3c signaling and thus contributes to the regulation of spermatogonial maintenance.

The neural cell adhesion molecule-derived peptide, FGL, attenuates lipopolysaccharide-induced changes in glia in a CD200-dependent manner.

Fibroblast growth loop (FGL) is a neural cell adhesion molecule (NCAM)-mimetic peptide that mimics the interaction of NCAM with fibroblast growth factor receptor (FGFR). FGL increases neurite outgrowth and promotes neuronal survival in vitro, and it has also been shown to have neuroprotective effects in vivo. More recent evidence has indicated that FGL has anti-inflammatory effects, decreasing age-related changes in microglial activation and production of inflammatory cytokines. These changes have been associated with an FGL-induced increase in expression of the glycoprotein, CD200, which interacts with its receptor to help maintain microglia in a quiescent state. However whether the FGL-induced anti-inflammatory effects are CD200-dependent has not been examined. The objective of this study was to address this question. Mixed glia were prepared from brain tissue of neonatal wildtype and CD200-deficient mice and preincubated with FGL prior to stimulation with lipopolysaccharide (LPS). Cells were assessed for mRNA expression of markers of microglial activation, CD11b, CD40 and intercellular adhesion molecule 1 (ICAM-1) and also the inflammatory cytokines, interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α, while supernatant concentrations of these cytokine were also assessed. LPS significantly increased all these parameters and the effect was greater in cells prepared from CD200-deficient mice. Whereas FGL attenuated the LPS-induced changes in cells from wildtype mice, it did not do so in cells from CD200-deficient mice. We conclude that the FGL-induced changes in microglial activation are CD200-dependent and demonstrate that the interaction of astrocytes with microglia is critically important for modulating microglial activation.

[Molecular genetic characteristics of the newcastle disease virus velogenic strains isolated in Russia, Ukraine, Kazakhstan, and Kirghizia].

The F gene fragment of 79 Newcastle disease virus (NDV) strains isolated from domestic and synanthropic birds in Kazakhstan, Kirghizia, Ukraine, and Russia in 1993 to 2007 was comparatively analyzed. Phylogenetic analysis of test isolates and reference NDV strains obtained from the GenBank was carried out by polymerase chain reaction with subsequent sequencing and comparative analysis of 154-bp nucleotide sequences in the main functional region of the F gene. All newly characterized isolates belong to three NDV genotype VII subgroups: VIIa, VIIb, VIId. The results show it necessary to monitor of NDV strains isolated in the CIS countries since the spread of NDV among migratory and synanthropic birds (pigeons, crows, and jackdaws) poses a serious threat to commercial poultry industry.

Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens.

Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen concentration of ISCOMs, containing Eimeria tenella antigens and saponins from native plants, were evaluated in their ability to stimulate humoral immunity and to protect chickens against a challenge infection with E. tenella. Broiler chickens were immunized with ISCOM preparations containing E. tenella antigens and the purified saponins Gg6, Ah6 and Gp7 isolated from Glycyrrhiza glabra, Aesculus hippocastanum and Gipsophila paniculata, respectively. The effects of the route of administration, dose of antigen and type of saponin used for construction of ISCOMs were evaluated for ability to stimulate serum IgG and IgM and to protect chickens against a homologous challenge. A single intranasal immunization was the most effective route for administering ISCOMs although the in ovo route was also quite effective. Dose titration experiments demonstrated efficacy after single immunization with various ISCOM doses but maximum effects were observed when ISCOMs contain 5-10mug antigen. Immunization of birds by any of the three routes with E. tenella antigens alone or antigens mixed with alum hydroxide adjuvant resulted in lower serum antibody and reduced protection to challenge relative to immunization with ISCOMs. Overall the results of this study confirm that significant immunostimulation and protection to challenge are achieved by immunization of chickens with ISCOMs containing purified saponins and native E. tenella antigens and suggest that ISCOMs may be successfully used to develop a safe and effective vaccine for prevention of avian coccidiosis.

Signaling mechanisms of neurite outgrowth induced by the cell adhesion molecules NCAM and N-cadherin.

Formation of appropriate neural circuits depends on a complex interplay between extracellular guiding cues and intracellular signaling events that result in alterations of cytoskeletal dynamics and a neurite growth response. Surface-expressed cell adhesion molecules (CAMs) interact with the surroundings via the extracellular domain and bind to the cytoskeleton via their intracellular domain. In addition, several CAMs induce signaling events via direct interactions with intracellular proteins or via interactions with cell surface receptors. Thus, CAMs are obvious candidates for transmitting extracellular guidance cues to intracellular events and thereby regulating neurite outgrowth. In this review, we focus on two CAMs, the neural cell adhesion molecule (NCAM) and N-cadherin, and their ability to mediate signaling associated with a neurite outgrowth response. In particular, we will focus on direct interaction between NCAM and N-cadherin with a number of intracellular partners, as well as on their interaction with the fibroblast growth factor receptor (FGFR).

Dimerization effect of sucrose octasulfate on rat FGF1.

Fibroblast growth factors (FGFs) constitute a family of at least 23 structurally related heparin-binding proteins that are involved in regulation of cell growth, survival, differentiation and migration. Sucrose octasulfate (SOS), a chemical analogue of heparin, has been demonstrated to activate FGF signalling pathways. The structure of rat FGF1 crystallized in the presence of SOS has been determined at 2.2 A resolution. SOS-mediated dimerization of FGF1 was observed, which was further supported by gel-filtration experiments. The major contributors to the sulfate-binding sites in rat FGF1 are Lys113, Lys118, Arg122 and Lys128. An arginine at position 116 is a consensus residue in mammalian FGF molecules; however, it is a serine in rat FGF1. This difference may be important for SOS-mediated FGF1 dimerization in rat.

The NCAM-derived P2 peptide facilitates recovery of cognitive and motor function and ameliorates neuropathology following traumatic brain injury.

The neural cell adhesion molecule (NCAM) plays a crucial role during development and regeneration of the nervous system, mediating neuronal differentiation, survival and plasticity. Moreover, NCAM regulates learning and memory. A peptide termed P2, corresponding to a 12-amino-acid sequence in the second immunoglobulin (Ig)-like module of NCAM, represents the natural cis-binding site for the first NCAM Ig module. The P2 peptide targets NCAM, thereby inducing a number of intracellular signaling events leading to the stimulation of neurite outgrowth and promotion of neuronal survival in vitro. The present study evaluated the effect of the P2 peptide on functional and histological outcomes following traumatic brain injury inflicted by a cortical cryogenic lesion. Lesioned rats were injected subcutaneously with P2 peptide, 5 mg/kg daily for 15 days beginning 2 h after injury. This treatment significantly improved postlesion recovery of motor and cognitive function, reduced neuronal degeneration, protected cells against oxidative stress, and increased reactive astrogliosis and neuronal plasticity in the sublesional area. P2 appeared rapidly in blood and cerebrospinal fluid after subcutaneous administration and remained detectable in blood for up to 5 h. The results suggest that P2 has therapeutic potential for the treatment of traumatic brain injury.

Immunostimulating complexes incorporating Eimeria tenella antigens and plant saponins as effective delivery system for coccidia vaccine immunization.

Immunostimulating complexes (ISCOMs) are unique, multimolecular structures formed by encapsulating antigens, lipids, and triterpene saponins of plant origin, and are an effective delivery system for various kinds of antigens. The uses of ISCOMs formulated with saponins from plants collected in Kazakhstan, with antigens from the poultry coccidian parasite Eimeria tenella, were evaluated for their potential use in developing a vaccine for control of avian coccidiosis. Saponins isolated from the plants Aesculus hippocastanum and Glycyrrhiza glabra were partially purified by HPLC. The saponin fractions obtained from HPLC were evaluated for toxicity in chickens and chicken embryos. The HPLC saponin fractions with the least toxicity, compared to a commercial saponin Quil A, were used to assemble ISCOMs. When chicks were immunized with ISCOMs prepared with saponins from Kazakhstan plants and E. tenella antigens, and then challenged with E. tenella oocysts, significant protection was conveyed compared to immunization with antigen alone. The results of this study indicate that ISCOMs formulated with saponins isolated from plants indigenous to Kazakhstan are an effective antigen delivery system which may be successfully used, with low toxicity, for preparation of highly immunogenic coccidia vaccine.

Effects of P2, a peptide derived from a homophilic binding site in the neural cell adhesion molecule on learning and memory in rats.

The neural cell adhesion molecule (NCAM) plays a pivotal role in neural development, regeneration, synaptic plasticity, and memory processes. P2 is a 12-amino-acid peptide derived from the second immunoglobulin-like (Ig) module of NCAM mediating cis-homophilic interactions between NCAM molecules present on the same cell. P2 is a potent NCAM agonist, capable of promoting neuronal differentiation and survival in vitro. The aim of this study was to assess the effect of P2 on learning and memory. Rats treated with P2 intracerebroventricularly (1 h prior to test) performed significantly better than controls in the reinforced T-maze, a test of spatial working memory. Further, rats treated with P2 exhibited decreased anxiety-like behavior while learning the T-maze task. In the social recognition test, both intracerebroventricular (1 h prior to test) and systemic (1 and 24 h prior to test) P2 treatment enhanced short-term social memory and counteracted (administration 24 h prior test) scopolamine-induced social memory impairment. In contrast, P2 (1 h prior to test) did not significantly improve long-term (24 h) retention of social memory, nor did it have any significant effects on long-term memory evaluated by the Morris water maze (administration between 2 days before training and 5.5 h posttraining). In the open field test, P2 (1 h prior to test) decreased general locomotion and rearing, but did not influence any other anxiety-related behaviors, indicating only a minimal influence on baseline anxiety levels. Taken together, these data indicate that in vivo P2 enhances short-term memory and protects against the amnestic effects of scopolamine, while modulating emotional behavior in a learning or novelty-related task.

A neural cell adhesion molecule-derived peptide reduces neuropathological signs and cognitive impairment induced by Abeta25-35.

By means of i.c.v. administration of preaggregated oligomeric beta-amyloid (Abeta)25-35 peptide it was possible in rats to generate neuropathological signs related to those of early stages of Alzheimer's disease (AD). Abeta25-35-administration induced the deposition of endogenously produced amyloid protein. Furthermore, quantitative immunohistochemistry demonstrated time-related statistically significant increases in amyloid immunoreactivity, tau phosphorylation, microglial activation, and astrocytosis, and stereological investigations demonstrated statistically significant increased neuronal cell death and brain atrophy in response to Abeta25-35. Finally, the Abeta25-35-administration led to a reduced short-term memory as determined by the social recognition test. A synthetic peptide termed FGL derived from the neural cell adhesion molecule (NCAM) was able to prevent or, if already manifest, strongly reduce all investigated signs of Abeta25-35-induced neuropathology and cognitive impairment. The FGL peptide was recently demonstrated to be able to cross the blood-brain-barrier. Accordingly, we found that the beneficial effects of FGL were achieved not only by intracisternal, but also by intranasal and s.c. administration of the peptide. Furthermore, FGL-treatment was shown to inhibit the activity of GSK3beta, a kinase implicated in signaling regulating cell survival, tau phosphorylation and the processing of the amyloid precursor protein (APP). Thus, the peptide induced a statistically significant increase in the fraction of GSK3beta phosphorylated on the Ser9-position, a posttranslational modification known to inhibit the activity of the kinase. Hence, the mode of action of FGL with respect to the preventive and curative effects on Abeta25-35-induced neuropathological manifestations and cognitive impairment involves the modulation of intracellular signal-transduction mediated through GSK3beta.

A neural cell adhesion molecule-derived fibroblast growth factor receptor agonist, the FGL-peptide, promotes early postnatal sensorimotor development and enhances social memory retention.

The neural cell adhesion molecule (NCAM) belongs to the immunoglobulin (Ig) superfamily and is composed extracellularly of five Ig-like and two fibronectin type III (F3) modules. It plays a pivotal role in neuronal development and synaptic plasticity. NCAM signals via a direct interaction with the fibroblast growth factor receptor (FGFR). A 15-amino-acid long peptide, the FG loop (FGL) peptide, that is derived from the second F3 module of NCAM has been found to activate FGFR1. We here report that the FGL peptide, when administered intranasally to newborn rats, accelerated early postnatal development of coordination skills. In adult animals s.c. administration of FGL resulted in a prolonged retention of social memory. We found that FGL rapidly penetrated into the blood and cerebrospinal fluid after both intranasal and s.c. administration and remained detectable in the fluids for up to 5 hours.

IL-1beta-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line.

AIMS/HYPOTHESIS: IL-1beta released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1beta-induced pro-apoptotic signalling in beta cells. Pancreatic beta cells express NCAM, but its biological effects in these cells are unclear. The aim of this study was to investigate whether there is cross-talk between NCAM signalling and cytokine-induced pro-apoptotic signalling. MATERIALS AND METHODS: Western blotting was used to investigate levels of NCAM and inducible nitric oxide synthase, phosphorylation of Src and MAPKs, and cleavage of caspase-3. MAPK activity was investigated with an in vitro kinase assay. Apoptosis was detected by cleaved caspase-3 and a Cell Death Detection ELISA(plus) assay. NCAM-induced fibroblast growth factor receptor (FGFR) activation was investigated in NCAM(-/-) Trex293 cells where FGFR phosphorylation was measured by Western blotting after NCAM transfection. RESULTS: Pre-exposure of INS-1E cells to the FGFR-inhibitor SU5402, but not to the Src-inhibitor PP2, dose-dependently inhibited IL-1beta-mediated MAPK activity. A synthetic peptide, C3d, reported to bind NCAM, did not activate MAPK or Akt as reported in neurons but inhibited IL-1beta-induced MAPK activity, thereby mimicking the effect of SU5402. Furthermore, C3d inhibited NCAM-induced FGFR phosphorylation and apoptosis induced by IL-1beta plus IFN-gamma, but did not affect IL-1beta-induced NF-kappaB signalling. CONCLUSIONS/INTERPRETATION: We suggest that NCAM signalling through FGFR is required for efficient IL-1beta pro-apoptotic signalling by facilitating IL-1beta-induced MAPK activation downstream of the NF-kappaB-MAPK branching point. Further, these data identify a novel function of C3d as an inhibitor of NCAM-induced FGFR activity and of IL-1beta-induced MAPK activation in beta cells.

Cell-cycle-dependent regulation of cell motility and determination of the role of Rac1.

To study cell motility in different phases of the cell cycle, time-lapse recording by computer-assisted microscopy of unsynchronised cells from three mammalian cell lines (L929, BT4Cn, HeLa) was used for the determination of the displacements of individual cells. The displacements were used for calculation of three key parameters describing cell motility: speed, persistence time and rate of diffusion. All investigated cell lines demonstrated a lower cell displacement in the G2 phase than in the G1/S phases. This was caused by a decrease in speed and/or persistence time. The decrease in motility was accompanied by changes in morphology reflecting the larger volume of cells in G2 than in G1. Furthermore, L-cells and HeLa-cells appeared to be less adherent in the G2 phase. Transfection of L-cells with constitutively active Rac1 led to a general increase in the speed and rate of diffusion in G2 to levels comparable to those of control cells in G1. In contrast, transfection with dominant-negative Rac1 reduced cell speed and resulted in cellular displacements, which were identical in G1 and G2. These observations indicate that migration of cultured cells is regulated in a cell-cycle-dependent manner, and that an enhancement of Rac1 activity is sufficient for a delay of the reduced cell displacement otherwise seen in G2.

Post-training administration of a synthetic peptide ligand of the neural cell adhesion molecule, C3d, attenuates long-term expression of contextual fear conditioning.

The neural cell adhesion molecule (NCAM) plays a key role in synaptic plasticity and memory formation. We have recently developed a synthetic peptide, termed C3d, which, through the binding to the first, N-terminal immunoglobulin-like (Ig) module in the extracellular portion of NCAM, has been shown to promote neurite outgrowth and synapse formation in vitro, and to interfere with passive avoidance memory in rats in vivo. In this study, we investigated whether the i.c.v. administration of C3d, either 5.5 h after or 2 days before training, could be effective to modulate the strength at which emotional memory for aversive situations is established into a long-term memory. The effects of the peptide were evaluated in adult male Wistar rats trained in the contextual fear conditioning task. The results indicated that C3d significantly reduced the subsequent long-term retention of the conditioned fear response when administered 5.5 h post-training, as indicated by retention tests performed 2-3 and 7 days post-training. However, this treatment failed to influence conditioning for this task when injected 2 days pre-training. Additional experiments showed that C3d did not influence the emotional or locomotor behaviour of the animals, when tested in the open field task. Furthermore, hippocampal levels of microtubule-associated protein 2 (MAP2), Synaptophysin and NCAM were found unchanged when evaluated by enzyme-linked immunosorbent assay in crude synaptosomal preparations 2 days after peptide i.c.v. injection. Therefore, post-training injection of this synthetic peptide was efficient to attenuate the strength at which memory for contextual fear conditioning was enduringly stored, whilst it did not affect the acquisition of new memories. In addition to further support the view that NCAM is critically involved in memory consolidation, the current findings suggest that the NCAM IgI module is a potential target for the development of therapeutic drugs capable to reduce the cognitive impact induced by exposure to intensive stress experiences.

[Mexidol-induced modification of lipid metabolism in pancreatitis]. / Modifikatsiia obmena lipidov pri pankreatite pod vliianiem meksidola.

The chronic experiments on mongrel dogs with a model pancreatitis showed that mexidol decreases manifestations of the inflammatory process. The treatment with mexidol led to a decrease in the degree of lipid transformations in the initial stage of pancreatitis development, with normaliation of the lipid metabolism according to the liver and blood plasma characteristics. The membranoprotector effect of mexidol, manifested in normalization of the lipid spectrum, is probably related to inhibition of the lipid peroxidation (LPO) process and to a decrease in the activity of phospholipase A2. The correlation between lipid metabolism, LPO, and phospholipase A2 activity in the tissues studied indicates that the therapeutic effect of mexidol in animals with pancreatitis is based on the cytoprotector activity of the drug.

[Dimephosphon in complex therapy of acute edematous pancreatitis]. / Dimefosfon v kompleksnoi terapii ostrogo otechnogo pankreatita.

Clinical investigations of 120 patients have shown that Dimephosphon included in complex treatment of acute edematous pancreatitis facilitates more rapid arrest of the symptoms. An important effect of the drug is its ability to decrease the level of endogenous intoxication by both the hydrophilic and hydrophobic components. It was established that one of the mechanisms of favorable action of the drug is its ability to inhibit LPO, to decrease activity of phospholipase A2. The drug possessing the cytoprotecting effect promotes rapid restoration of the functional state of the liver, in particular its detoxicating, albumin synthesizing ability that is not least of the factors of optimization of the complex therapy.