Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles.

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-ß-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20-30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

Heating-induced transition of Potyvirus Potato Virus A coat protein into ß-structure.

In our previous communication, we have reported that virions of plant Potyvirus Potato Virus A (PVA) have a peculiar structure characterized by high content of disordered regions in intravirus coat protein (CP). In this report, we describe unusual properties of the PVA CP. With the help of a number of physicochemical methods, we have observed that the PVA CP just released from the virions by heating at 60-70 °C undergoes association into oligomers and transition to ß- (and even cross-ß-) conformation. Transition to ß-structure on heating has been recently reported for a number of viral and non-viral proteins. The PVA CP isolated by LiCl method was also transformed into cross-ß-structure on heating to 60 °C. Using the algorithms for protein aggregation prediction, we found that the aggregation-prone segments should be located in the central region of a PVA CP molecule. Possibly this transition mimics some functions of PVA CP in the virus life cycle in infected plants.

Partially disordered structure in intravirus coat protein of potyvirus potato virus A.

Potyviruses represent the most biologically successful group of plant viruses, but to our knowledge, this work is the first detailed study of physicochemical characteristics of potyvirus virions. We measured the UV absorption, far and near UV circular dichroism spectra, intrinsic fluorescence spectra, and differential scanning calorimetry (DSC) melting curves of intact particles of a potato virus A (PVA). PVA virions proved to have a peculiar combination of physicochemical properties. The intravirus coat protein (CP) subunits were shown to contain an unusually high fraction of disordered structures, whereas PVA virions had an almost normal thermal stability. Upon heating from 20 °C to 55 °C, the fraction of disordered structures in the intravirus CP further increased, while PVA virions remained intact at up to 55 °C, after which their disruption (and DSC melting) started. We suggest that the structure of PVA virions below 55 °C is stabilized by interactions between the remaining structured segments of intravirus CP. It is not improbable that the biological efficiency of PVA relies on the disordered structure of intravirus CP.

Aberrant glycosylation of the anti-Thomsen-Friedenreich glycotope immunoglobulin G in gastric cancer patients.

AIM: To study whether alterations in the glycosylation of immunoglobulin G (IgG) specific to the Thomsen-Friedenreich glycotope (TF) have diagnostic and prognostic potential in gastric cancer. METHODS: Serum samples were obtained from patients with histologically verified gastric carcinoma (n = 89), healthy blood donors (n = 40), and patients with benign stomach diseases (n = 22). The lectin-enzyme-linked immunosorbent assay-based glycoprofiling of TF-specific IgG (anti-TF IgG) was performed using synthetic TF-polyacrylamide conjugate as antigen, total IgG purified by affinity chromatography on protein G sepharose, and lectins of various sugar specificities: mannose-specific concanavalin A (ConA), fucose-specific Aleuria aurantia lectin (AAL) and sialic acid-specific Sambucus nigra agglutinin (SNA). The sensitivity and specificity of the differences between cancer patients and controls were evaluated by receiver operator characteristic (ROC) curve analysis. Overall survival was analyzed by the Kaplan-Meier method. Time-dependent ROC curve statistics were applied to determine cut-off values for survival analysis. All calculations and comparisons were performed using the GraphPad Prism 5 and SPSS 15.0 software. RESULTS: The level of TF-specific IgG was significantly increased in cancer patients compared with non-cancer controls (P < 0.001). This increase was pronounced mostly in stage 1 of the disease. Cancer patients showed a higher level of ConA binding to anti-TF-IgG (P < 0.05) and a very low level of SNA lectin binding (P = 0.0001). No appreciable stage-dependency of the binding of any lectin to anti-TF IgG was found. A strong positive correlation between the binding of AAL and SNA was found in all groups studied (r = 0.71-0.72; P < 0.0001). The changes in ConA reactivity were not related to those of the fucose- or sialic acid-specific lectin. Changes in the SNA binding index and the ConA/SNA binding ratio demonstrated good sensitivity and specificity for stomach cancer: sensitivity 78.79% (95%CI: 61.09-91.02) and 72.73% (95%CI: 57.21-85.04); specificity 79.17 (95%CI: 65.01-89.53) and 88.64% (95%CI: 71.8-96.6), for the SNA binding index and the ConA/SNA binding ratio, respectively. The other combinations of lectins did not improve the accuracy of the assay. The low level of ConA-positive anti-TF IgG was associated with a survival benefit in cancer patients (HR = 1.56; 95%CI: 0.78-3.09; P = 0.19), especially in stages 3-4 of the disease (HR = 2.17; 95%CI: 0.98-4.79; P = 0.048). A significantly better survival rate was found in all cancer patients with a low reactivity of anti-TF IgG to the fucose-specific AAL lectin (HR = 2.39; 95%CI: 1.0-5.7; P = 0.038). CONCLUSION: The changes in the TF-specific IgG glycosylation pattern can be used as a biomarker for stomach cancer detection, and to predict patient survival.

Tick-borne pathogens in ticks feeding on migratory passerines in Western part of Estonia.

During southward migration in the years 2006-2009, 178 migratory passerines of 24 bird species infested with ticks were captured at bird stations in Western Estonia. In total, 249 nymphal ticks were removed and analyzed individually for the presence of Borrelia burgdorferi sensu lato (s.l.), tick-borne encephalitis virus (TBEV), and Anaplasma phagocytophilum. The majority of ticks were collected from Acrocephalus (58%), Turdus (13%), Sylvia (8%), and Parus (6%) bird species. Tick-borne pathogens were detected in nymphs removed from Acrocephalus, Turdus, and Parus bird species. TBEV of the European subtype was detected in 1 I. ricinus nymph removed from A. palustris. B. burgdorferi s.l. DNA was found in 11 ticks (4.4%) collected from Turdus and Parus species. Bird-associated B. garinii and B. valaisiana were detected in I. ricinus nymphs removed from T. merula. Rodent-associated B. afzelii was detected in 3 I. ricinus nymphs from 2 P. major birds. One of the B. afzelii-positive nymphs was infected with a mix of 2 B. afzelii strains, whereas 1 of these strains was also detected in another nymph feeding on the same great tit. The sharing of the same B. afzelii strain by 2 nymphs indicates a possible transmission of B. afzelii by co-feeding on a bird. A. phagocytophilum DNA was detected in 1 I. ricinus nymph feeding on a T. iliacus. The results of the study confirm the possible role of migratory birds in the dispersal of ticks infected with tick-borne pathogens along the southward migration route via Estonia.

Detection and genetic characterization of relapsing fever spirochete Borrelia miyamotoi in Estonian ticks.

During the years 2008-2010 I. ricinus and I. persulcatus ticks were collected from 64 sites in mainland Estonia and on the island Saaremaa. Presence of B. miyamotoi was found in 0.9% (23/2622) of ticks. The prevalence in I. persulcatus and I. ricinus ticks differed significantly, 2.7% (15/561) and 0.4% (8/2061), respectively. The highest prevalence rates were in found South-Eastern Estonia in an area of I. persulcatus and I. ricinus sympatry and varied from 1.4% (1/73) to 2.8% (5/178). Co-infections with B. burgdorferi s.l. group spirochetes and tick-borne encephalitis virus were also revealed. Genetic characterization of partial 16S rRNA, p66 and glpQ genes demonstrated that Estonian sequences belong to two types of B. miyamotoi and cluster with sequences from Europe and the European part of Russia, as well as with sequences from Siberia, Asia and Japan, here designated as European and Asian types, respectively. Estonian sequences of the European type were obtained from I. ricinus ticks only, whereas the Asian type of B. miyamotoi was shown for both tick species in the sympatric regions.

Molecular diversity of snake venom nerve growth factors.

Nerve growth factor (NGF) is a protein which stimulates the differentiation and maintenance of sympathetic and embryonic sensory neurons. Snake venoms are a rich source of NGF. Due to small quantities it is sometimes difficult and laborious to isolate NGF from the venoms. In this study the use of Ni-NTA-agarose for isolation of NGF is studied. Anti-Vipera lebetina NGF antibodies were used for identification of NGF during Ni-NTA-agarose fractionation as well as for cross-reaction studies with 21 snake venoms. All studied venoms contained NGF. The molecular masses of the NGFs from Echis ocellatus, Agkistrodon contortrix contortrix, A. bilineatus, A. blomhoffii, A. saxatilis, Calloselasma rhodostoma, Bothrops jararaca and B. lanceolatus were determined for the first time. Some previous results of the NGF studies are revaluated.

Detection and characterization of Babesia species in Ixodes ticks in Estonia.

The presence of Babesia spp. was studied in 2603 Ixodes ricinus and Ixodes persulcatus ticks collected at seven sites in Estonia. By reverse line blot screening, Babesia spp. was detected in 36 (1.4%) ticks, among them 18 (0.7%) were further recognized by a Babesia microti probe, 3 (0.1%) by a Babesia divergens probe, and the other 15 (0.6%) were recognized only by the universal Babesia spp. "catch all" probe. Sequence analyses of 6 of these 15 samples revealed that all of them belonged to Babesia sp. EU1. B. microti was detected in both tick species I. ricinus and I. persulcatus at the seven sites, whereas B. divergens-like and Babesia sp. EU1 were found only in I. persulcatus and I. ricinus, respectively. Genetic characterization based on partial 18S rRNA showed that the Estonian sequences of B. microti, B. divergens-like, and Babesia sp. EU1 share a high rate of similarity and are closely related to sequences from other European countries, Siberia, and United States. The present study demonstrated for the first time the existence and distribution of Babesia spp. in I. persulcatus and I. ricinus ticks in Estonia.

Sonic Hedgehog pathway activity in prostate cancer.

Abnormal activation of the Sonic hedgehog (Shh) signaling pathway has been demonstrated in a number of human tumors, including prostate cancer. The study aimed to assess the activity of Shh pathway components (Shh, Gli1, Gli2 and Gli3), as well as the proliferation markers FoxA1 and Notch1 during cancer progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP). We evaluated changes in respective proteins by immunohistochemistry at three time points (12, 17 and 21 weeks of age) in the tissue of TRAMP and C57Bl/6 mice. Moreover, the expression of mRNA of these proteins was assessed. The present study shows a significant age-dependent increase in the number of Shh, Gli1, Gli3 and FoxA1-positive prostate cells and a decrease in Gli2-positive cells in TRAMP. The study also supports the hypothesis that the development of prostate cancer and its metastasis is associated with activation of the Shh signaling pathway.

Regulator of G protein signalling 16 is a target for a porcine circovirus type 2 protein.

Interaction studies have suggested that the non-structural protein encoded by open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) binds specifically to a regulator of G protein signalling (RGS) related to human RGS16 (huRGS16). The full-length clone of RGS16 was generated from porcine cells and sequence analysis revealed a close relationship to huRGS16 and murine RGS16. In vitro pull-down experiments verified an interaction between porcine RGS16 (poRGS16) and ORF3 from PCV2. Using GST-linked ORF3 proteins from three different genogroups of PCV2 and from porcine circovirus type 1 (PCV1) in the pull-down experiments indicated that there were differences in their ability to bind poRGS16. Quantitative RT-PCR demonstrated that the expression of poRGS16 mRNA could be induced by a number of cell activators including mitogens (LPS and PHA), interferon inducers (ODN 2216 and poly I : C) and the neurotransmitter norepinephrine. Immunofluorescence labelling confirmed the induced expression of poRGS16 at the protein level and suggested that the PCV2 ORF3 protein co-localized with poRGS16 in LPS-activated porcine PBMC. Furthermore, poRGS16 appeared to participate in the translocation of the ORF3 protein into the cell nucleus, suggesting that the observed interaction may play an important role in the infection biology of porcine circovirus.

Nerve growth factor from Vipera lebetina venom.

Nerve growth factor was isolated from the Vipera lebetina venom by a four-step procedure including gel filtration, ion exchange, heparin and hydrophobic chromatography. The purified protein is a glycosylated non-covalently bound homodimer with monomeric molecular mass of 14,380 Da. The cDNA encoding NGF is cloned and sequenced. The amino acid sequence translated from the cDNA comprises 117 or 119 amino acids depending on the N-terminus (truncated or not). The recombinant NGF (expressed in Escherichia coli) was used to prepare the anti-NGF antiserum. The antiserum interacted with the wild-type NGF and enabled to localize NGF during the purification procedure in parallel with MALDI-TOF analysis of tryptic peptides. The isolated NGF caused neurite outgrowth from PC12 cells in concentrations beginning from 2.5 ng/ml.

Generation and characterization of a single-chain Fv antibody against G, a hedgehog signaling pathway transcription factor.

Gli3 is a key regulator of development, controlling multiple patterning steps. Here we report the generation of a scFv antibody specific to the repressor domain of human Gli3. We show that this scFv retains the binding capacity of its parent anti-Gli3 monoclonal antibody derived from hybridoma clone 5E1. When expressed in mammalian cells, the anti-Gli3 scFv co-localizes with intracellular Gli3. Immunocytochemical staining of the intrabody in Gli3-positive TM4 cells shows a distinct perinuclear cytoplasmic localization. Such a scFv constitutes a useful tool for studying transcriptional regulation of the hedgehog pathway in mammals and offers a starting point for developing novel Gli-related therapeutic intrabodies.

Generation and characterization of mouse monoclonal antibody 5E1 against human transcription factor GLI3.

GLI3 is a transcriptional effector of the developmentally important hedgehog (Hh) signaling pathway. Here we report the production of mouse monoclonal antibody (MAb) against putative repressive motif in GLI3 (GLI3pRM). BALB/c mice were immunized with purified recombinant human GLI3pRM protein, and the splenocytes from these mice were fused with myeloma cell line (SP2/0) by using standard hybridoma production techniques. Resulting hybridomas producing anti-GLI3pRM antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. The specificity of MAb 5E1 was determined based on its activity in Western blot and immunofluorescence analyses of the human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/kappa, recognizing recombinant human GLI3pRM specifically. In addition, MAb 5E1 bound to the full-length (FL-GLI3) as well as a short protein (GLI3R) and did not cross-react with a similar region in GLI2. MAb 5E1 could also be used to detect the expression of Gli3 in mouse cell lines and embryonic tissues.

Generation and characterization of mouse monoclonal antibody 5E1 against human transcription factor GLI3.

GLI3 is a transcriptional effector of the developmentally important hedgehog (Hh) signaling pathway. Here we report the production of mouse monoclonal antibody (MAb) against putative repressive motif in GLI3 (GLI3pRM). BALB/c mice were immunized with purified recombinant human GLI3pRM protein; the splenocytes from these mice were fused with myeloma cell line (SP2/0) by using standard hybridoma production techniques. Resulting hybridomas producing anti-GLI3pRM antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. The specificity of MAb 5E1 was determined based on its activity in Western blot and immunofluorescence analysis of human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/ê and it recognized recombinant human GLI3pRM specifically. In addition, MAb 5E1 bound to the full-length (FL-GLI3) as well as a short protein (GLI3R) and did not cross-react with a similar region in GLI2. MAb 5E1 could also be used to detect the expression of GLI3 in mouse cell lines and embryonic tissues.

In vitro recombinants of two nearly identical potyviral isolates express novel virulence and symptom phenotypes in plants.

Six novel chimeric viruses were constructed by sequentially exchanging segments of the viral genomes between the infectious cDNA clone (pPVA-B11) of Potato virus A (isolate PVA-B11) and pUFL, an almost identical infectious cDNA of PVA (isolate U) made in this study. The infectious in vitro transcripts of pUFL and pPVA-B11 caused similar severe mosaic and leaf malformation phenotypes in systemically infected leaves of Nicotiana benthamiana. In contrast, one chimera induced a unique phenotype of yellow vein chlorosis without leaf malformation with viral titres that were equivalent to those of the parental viruses. Furthermore, as opposed to the viral cDNAs from which it was assembled, one chimera showed no detectable infectivity of N. benthamiana plants. Thus, recombination of nearly identical, phenotypically similar virus genomes can give rise to new viral strains with novel virulence and symptom phenotypes, which has not previously been demonstrated with potyviruses. One chimera failed to cause systemic infection in potato plants, but, nevertheless, avirulence could not be attributed to a single genomic region. These data suggest that different parts of the potyviral genome function coordinately. The results provide novel insights into the evolution of the genus Potyvirus.