Histopathology of <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> (Acari: Ixodidae) ticks fed on resistant hosts

Histological features of Rhipicephalus sanguineus ticks fed on dog, a non resistant host, and on guinea pig, a resistant host, were compared. Unfed ticks and ticks from each host species were collected during first and third infestation and processed for histology. Many ticks from guinea pigs, especially during third infestation, were unattached, dehydrated and small. Only the midgut of ticks fed on guinea pigs had host leukocytes. Vacuolization of midgut cells was observed in all ticks, with exception of those fed on dogs for more than 96 h. Ticks of guinea pigs, particularly from third infestation, had vacuolated tracheae and swelling of malpighian tubules. Solely ticks from third infestation of guinea pigs displayed vacuolization of oocytes. Ticks fed on guinea pigs also had an increased number of guanine spherules. Observed alterations in ticks from guinea pigs are discussed.     

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Aims: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease‐catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent‐tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. Methods and Results: The tripeptide Ac‐Phe‐Gly‐Phe‐NH₂ was synthesized using Ac‐Phe‐OEt and Gly‐Phe‐NH₂ substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1·5 or 0·5 mol l^?1 NaCl. Purification and identification of the peptide product was achieved by RP‐HPLC and ESI‐MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Conclusion: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. Significance and Impact of Study: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.     

The ruthenium complex <Emphasis Type="Italic">cis</Emphasis>-(dichloro)tetrammineruthenium(III) chloride induces apoptosis and damages DNA in murine sarcoma 180 cells

Ruthenium(III) complexes are increasingly attracting the interest of researchers due to their promising pharmacological properties. Recently, we reported that the cis-(dichloro)tetrammineruthenium(III) chloride compound has cytotoxic effects on murine sarcoma 180 (S-180) cells. In an effort to understand the mechanism responsible for their cytotoxicity, study we investigated the genotoxicity, cell cycle distribution and induction of apoptosis caused by cis-(dichloro)tetrammineruthenium(III) chloride in S-180 tumour cells. cis-(dichloro)tetrammineruthenium(III) chloride treatment induced significant DNA damage in S-180 cells, as detected by the alkaline comet assay. In the cell cycle analysis, cis-(dichloro)tetrammineruthenium(III) chloride caused an increase in the number of cells in G1 phase, accompanied by a decrease in the S and G2 phases after 24 h of treatment. In contrast, the cell cycle distribution of S-180 cells treated with cis-(dichloro)tetrammineruthenium(III) chloride for 48 h showed a concentration-dependent increase in the sub-G1 phase (indicating apoptosis), with a corresponding decrease in cells in the G1, S and G2 phases. In addition, cis-(dichloro)tetrammineruthenium(III) chloride treatment induced apoptosis in a time-dependent manner, as observed by the increased numbers of annexin V-positive cells. Taken together, these findings strongly demonstrate that DNA damage, cell cycle changes and apoptosis may correlate with the cytotoxic effects of cis-(dichloro)tetrammineruthenium(III) chloride on S-180 cells.     

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.     

Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes

Background  

The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor.