Absence of pollinator-mediated premating barriers in mixed-ploidy populations of <Emphasis Type="Italic">Gymnadenia conopsea</Emphasis> s.l. (Orchidaceae)

Polyploidy has played a key role in plant evolution and diversification. Despite this, the processes governing reproductive isolation among cytotypes growing in mixed-ploidy populations are still largely unknown. Theoretically, coexistence of diploid and polyploid individuals in sympatric populations is unlikely unless cytotypes are prezygotically isolated through assortative pollination. Here, we investigated the pre-mating barriers involved in the maintenance of three co-occurring cytotypes from the genus Gymnadenia (Orchidaceae): tetraploid and octoploid G. conopsea and tetraploid G. densiflora. We assessed differences in flowering phenology, floral morphology, and visual and olfactory cues, which could lead to assortative mating. Gas chromatography coupled with electroantennographic detection was used to identify scent compounds with physiological activity in the two main pollinators, Deilephila porcellus and Autographa gamma. The importance of olfactory cues was also assessed in the field by analysing the moths’ responses to the olfactory display of the plants, and by following the pollinator’s behaviour on artificial arrays. Our complex approach demonstrated that the coexistence of Gymnadenia cytotypes in mixed-ploidy populations was only partly explained by differences in floral phenology, as cytotypes with overlapping flowering (i.e., octoploid G. conopsea and tetraploid G. densiflora) might freely exchange pollen due to only 1 mm differences in spur lengths and the lack of assortative behaviour of pollinators. While floral colour among the cytotypes was similar, floral scent differed significantly. Though both pollinator species seemed to physiologically detect these differences, and the floral scent alone was sufficient to attract them, pollinators did not use this cue to discriminate the cytotypes in the field. The absence of pre-mating barriers among cytotypes, except partial temporal segregation, suggests the existence of other mechanisms involved in the cytotypes’ coexistence. The genetic differences in ITS sequences among cytotypes were used to discuss the cytotype’s origin.     

Antioxidant system activation by mercury in Pfaffia glomerata plantlets

Oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets grown in nutrient solution using sand as substrate. Thirty-day-old acclimated plants were treated for 9 days with four Hg levels (0, 1, 25 and 50 μM) in the substrate. Parameters such as growth, tissue Hg concentration, toxicity indicators (δ-aminolevulinic acid dehidratase, δ-ALA-D, activity), oxidative damage markers (TBARS, lipid peroxidation, and H₂O₂ concentration) and enzymatic (superoxide dismutase, SOD, catalase, CAT, and ascorbate peroxidase, APX) and non-enzymatic (non-protein thiols, NPSH, ascorbic acid, AsA, and proline concentration) antioxidants were investigated. Tissue Hg concentration increased with Hg levels. Root and shoot fresh weight and δ-ALA-D activity were significantly decreased at 50 μM Hg, and chlorophyll and carotenoid concentration were not affected. Shoot H₂O₂ concentration increased curvilinearly with Hg levels, whereas lipid peroxidation increased at 25 and 50 μM Hg, respectively, in roots and shoots. SOD activity showed a straight correlation with H₂O₂ concentration, whereas CAT activity increased only in shoots at 1 and 50 μM Hg. Shoot APX activity was either decreased at 1 μM Hg or increased at 50 μM Hg. Conversely, root APX activity was only increased at 1 μM Hg. In general, AsA, NPSH and proline concentrations increased upon addition of Hg, with the exception of proline in roots, which decreased. These changes in enzymatic and non-enzymatic antioxidants had a significant protective effect on P. glomerata plantlets under mild Hg-stressed conditions.     

Synthesis and pharmacological evaluation of pyrazine N-acylhydrazone derivatives designed as novel analgesic and anti-inflammatory drug candidates

In this paper, we report the synthesis and pharmacological evaluation of pyrazine N-acylhydrazone (NAH) derivatives (2a–s) designed as novel analgesic and anti-inflammatory drug candidates. This series was planned by molecular simplification of prototype 1 (LASSBio-1018), previously described as a non-selective cyclooxygenase inhibitor. Derivatives 2a–s were evaluated in several animal models of pain and inflammation, standing-out compound 2o (2-N′-[(E)-(3,4,5-trimethoxyphenyl) methylidene]-2-pyrazinecarbohydrazide; LASSBio-1181), that was also active in a murine model of chronic inflammation (i.e., adjuvant-induced arthritis test in rats) and can be considered a new analgesic and anti-inflammatory lead for drug development.     

An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes

Leptospira spp. serovars Hardjo and Wollfi from Sejroe serogroup have been detected in livestock in Brazil, where the main control procedures rely on vaccination. The potency of two commercial vaccines available in this country was monitored by microagglutination test-MAT and in vitro growth inhibition test-GIT in serum samples from 33 female buffaloes divided into: G1-unvaccinated control; G2-vaccinated with Leptobac-6^® containing serovars Hardjo and Wolffi and G3-vaccinated with Triangle-9^® containing serovar Hardjo. G2 and G3 animals were vaccinated on day zero, and received a booster and two revaccinations on days 30, 210 and 390 and G1 animals received phosphate buffered saline. Serum samples were collected at 15-day intervals between days 0 and 60; and at 30-day intervals between days 60 and 540 and were tested by MAT and GIT with serovars Hardjo and Wolffi. G1 remained negative throughout the experiment. Both vaccines were able to induce agglutinating and growth inhibition antibodies. Six months after the last revaccination, all animals tested negative by MAT, but still were positive by GIT until the end of experimental period. GIT could be a good tool to evaluate the potency and to monitor antibodies responses of vaccines of Sejroe group serovars.     

A combined RT‐PCR and dot‐blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius)

Aims: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red‐spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. Methods and Results: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin‐labelled probes resulted in the identification of both genotypes separately. The application of the RT‐PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46·87% of viral nervous necrosis virus carriers. The combination of RT‐PCR and blot hybridization increases the detection rate up to 90·62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56·25%). Conclusions: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. Significance and Impact of the Study: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.     

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin‐conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti‐intimin IgG‐enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1·3 × 10^?8 mol l^?1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae‐negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti‐intimin IgG‐enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti‐intimin IgG‐enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.     

Young patients with colorectal cancer have poor survival in the first twenty months after operation and predictable survival in the medium and long-term: Analysis of survival and prognostic markers

Introduction

Male breast cancer (MBC) is a rare, yet potentially aggressive disease. Although literature regarding female breast cancer (FBC) is extensive, little is known about the etiopathogenesis of male breast cancer. Studies from our laboratory show that MBCs have a distinct immunophenotypic profile, suggesting that the etiopathogenesis of MBC is different from FBCs. The aim of this study was to evaluate and correlate the immunohistochemical expression of cell cycle proteins in male breast carcinoma to significant clinico-biological endpoints.

Methods

75 cases of MBC were identified using the records of the Saskatchewan Cancer Agency over 26 years (1970-1996). Cases were reviewed and analyzed for the immunohistochemical expression of PCNA, Ki67, p27, p16, p57, p21, cyclin-D1 and c-myc and correlated to clinico-biological endpoints of tumor size, node status, stage of the disease, and disease free survival (DFS).

Results

Decreased DFS was observed in the majority of tumors that overexpressed PCNA (98%, p = 0.004). The overexpression of PCNA was inversely correlated to the expression of Ki67 which was predominantly negative (78.3%). Cyclin D1 was overexpressed in 83.7% of cases. Cyclin D1 positive tumors were smaller than 2 cm (55.6%, p = 0.005), had a low incidence of lymph node metastasis (38.2%, p = 0.04) and were associated with increased DFS of >150 months (p = 0.04). Overexpression of c-myc (90%) was linked with a higher incidence of node negativity (58.3%, p = 0.006) and increased DFS (p = 0.04). p27 over expression was associated with decreased lymph node metastasis (p = 0.04). P21 and p57 positive tumors were related to decreased DFS (p = 0.04). Though p16 was overexpressed in 76.6%, this did not reach statistical significance with DFS (p = 0.06) or nodal status (p = 0.07).

Conclusion

Aberrant cell cycle protein expression supports our view that these are important pathways involved in the etiopathogenesis of MBC. Tumors with overexpression of Cyclin D1 and c-myc had better outcomes, in contrast to tumors with overexpression of p21, p57, and PCNA with significantly worse outcomes. P27 appears to be a predictive marker for lymph nodal status. Such observation strongly suggests that dysregulation of cell cycle proteins may play a unique role in the initiation and progression of disease in male breast cancer. Such findings open up new avenues for the treatment of MBC as a suitable candidate for novel CDK-based anticancer therapies in the future.     

Mutational analysis reveals a complex interplay of peptide binding and multiple biological features of HLA-B27

Molecular polymorphism influences the strong association of HLA-B27 with ankylosing spondylitis through an unknown mechanism. Natural subtypes and site-directed mutants were used to analyze the effect of altering the peptide-binding site of this molecule on its stability, interaction with tapasin, folding, and export. The disease-associated subtypes B*2705, B*2702, and B*2704 showed higher thermostability at 50 °C than all other subtypes and mutants, except some mimicking B*2702 polymorphism. The lowest values were found among pocket B mutants, most of which interacted strongly with tapasin, but otherwise there was no correlation between thermostability and tapasin interaction. Mutants resulting in increased hydrophobicity frequently acquired their maximal thermostability faster than those with increased polarity, suggesting that this process is largely driven by the thermodynamics of peptide binding. Folding, export, and tendency to misfold were influenced by polymorphism all along the peptide-binding site and were not specifically dependent on any particular region or structural feature. Frequent uncoupling of thermostability, folding/misfolding, and export can be explained by the distinct effect of mutations on the acquisition of a folded conformation, the optimization rate of B27-peptide complexes, and their quality control in the endoplasmic reticulum, all of which largely depend on the ways in which mutations alter peptide binding, without excluding additional effects on interactions with tapasin or other proteins involved in folding and export. The similarity of the generally disease-associated B*2707 to nondisease-associated subtypes in all the features analyzed suggests that molecular properties other than antigen presentation may not currently explain the relationship between HLA-B27 polymorphism and ankylosing spondylitis.     

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as FcϵRI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased FcϵRI-induced degranulation, nuclear factor for T cell activation and NFκB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.