Slight influence of the estrous cycle stage on the mucosal and systemic specific antibody response induced after vaginal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis in mice

Since the immune response appears to be variable according to the hormonal stage of the mammalian female, the aim of this study was to determine whether estrous cycle stage modifies the mucosal and systemic immune responses induced by intraperitoneal and vaginal immunization of mice with protoxin Cry1Ac. We tested the influence of three immunizations on the specific antibody response elicited at estrus and diestrus, that were the same estrous cycle stages at which the antigen was applied. Both intraperitoneal and vaginal immunization of mice with Cry1Ac either at estrus or diestrus induces specific antibody responses at serum, vagina and large intestine. The stage of the estrous cycle have little or non influence in the magnitude of the response induced, since only at serum the IgM was slightly higher at estrus than at diestrus by both routes. At the large intestine only the IgA response elicited via the intraperitoneal route changed, being higher at diestrus, whereas at the vagina IgA response induced did not change significantly due to the cycle stage. Present results suggest that Cry1Ac may be used as an antigen carrier as it can elicit antibody responses at systemic level and at several mucosal sites including the vagina that are not modified significantly throughout the reproductive cycle.     

Halocidin: a new antimicrobial peptide from hemocytes of the solitary tunicate,Halocynthia aurantium

From hemocytes of the tunicate Halocynthia aurantium we purified a new antimicrobial peptide named halocidin. The native peptide had a mass of 3443 Da and comprised two different subunits containing 18 amino acid residues (WLNALLHHGLNCAKGVLA) and 15 residues (ALLHHGLNCAKGVLA), which were linked covalently by a single cystine disulfide bond. Two different monomers were separately synthesized and used to make three additional isoforms (15 residue homodimer, 18 residue homodimer, heterodimer). In antimicrobial assays performed with synthetic peptides of halocidin, it was confirmed that congeners of the 18 residue monomer were more active than those of the 15 residue monomer against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa.     

Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

The 2a (polymerase) protein of cucumber mosaic virus (CMV) was shown to be phosphorylated both in vivo and in vitro. In vitro assays using 2a protein mutants and tobacco protein kinases showed that the 2a protein has at least three phosphorylation sites, one of which is located within the N-terminal 126 amino acid region. This region is essential and sufficient for interaction with the CMV 1a protein. When phosphorylated in vitro, the 2a protein N-terminal region failed to interact with the 1a protein. Since the 1a-2a interaction is essential for the replication of CMV, this suggests that phosphorylation of the N-terminal region of the 2a protein negatively modulates the interaction in vivo, and may have a regulatory role acting directly in viral infection.     

Green fluorescent protein-labeled recombinant fluobody for detecting the picloram herbicide

A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC50 value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).     

Selective action of acetogenin mitochondrial complex I inhibitors

Five annonaceous acetogenins, rolliniastatin-1 [structure: see text], rolliniastatin-2 [structure: see text], laherradurin [structure: see text], squamocin [structure: see text], annonacin [structure: see text], and rotenone as a reference, differing in their NADH oxidase inhibition activity, have been evaluated for antifeedant, insecticidal, trypanocidal and cytotoxic effects on insect, mammalian and tumor cells. All the test compounds were toxic to Leptinotarsa decemlineata, demonstrated selective cytotoxicity to insect Sf9 cells and a panel of tumor cell lines with the multidrug-resistant SW480 (P-glycoprotein+, Pgp+) being the most sensitive one. Compounds [structure: see text] and rotenone had post-ingestive effects against Spodoptera littoralis larvae while [structure: see text] and rotenone were active against Trypanosoma cruzi. Based on their biochemical properties (inhibition of the mitochondrial NADH oxidase activity), the in vivo effects of these compounds on S. littoralis and their cytotoxic effects on Sf9 and tumor cells were more predictable than their effect on T. cruzi and mammalian cells.     

J-binding protein increases the level and retention of the unusual base J in trypanosome DNA

The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base beta-d-glucosyl-hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J-binding protein (JBP) in T. brucei. The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild-type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5-hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein.     

Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors

The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.     

Development and validation of a liquid chromatographic method for Casiopeina IIIi in rat plasma

In order to measure changes in physiological CO concentrations in blood with good accuracy, a method was developed using gas chromatography with flame ionisation detection (250 degrees C). A nickel catalyst system was fitted to convert CO to methane at 375 degrees C after separation with a molecular sieve column at 35 degrees C. Helium was used as carrier at 30 ml/min. Porcine or human blood (400 microl) was sampled in gastight tubes and treated with sulfuric acid and saponin (800 microl). Accuracy was 1.4% and 1.5% (RSD), respectively. Precision was 2.8% (porcine blood). Limit of detection was 0.01 nmol/ml gas and limit of quantification 12 nmol/ml blood. Calibration was made in the interval 12-514 nmol/ml blood (corresponding to 0.1-6% COHb). Samples were stable for at least a month at +4 degrees C. This paper describes a method with high sensitivity and good accuracy, suitable for analysis of low CO concentrations.     

Interleukin-4 inhibits the vascular endothelial growth factor- and basic fibroblast growth factor-induced angiogenesis in vitro

The role of interleukin-4 (IL-4) in the inflammatory process has emerged recently. In this study, we investigated the effect of IL-4 on the angiogenic process in an in vitro experimental system. IL-4 significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) that was induced by the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF- or bFGF-induced HUVEC chemotaxis was abrogated by the IL-4 treatment. In addition, the formation of tube-like structures by HUVEC in the presence of VEGF or bFGF was also severely down-regulated by IL-4. The inhibitory effects on the critical steps of angiogenesis were not observed with IL-6 that is abundantly found in the inflamed tissue. Our results suggest that IL-4 may play a regulatory role in normal physiology and provide the potential possibility for IL-4 as a therapeutic agent in the intervention of angiogenesis-related diseases.     

Juvenile-hormone-dependent interaction of actin and spectrin is crucial for polymorphic differentiation of the larval honey bee ovary

Programmed cell death in the worker ovary of Apis mellifera reduces the number of ovarioles during metamorphosis from 150-200 primordia to less than 10. In contrast, practically all ovarioles in the ovary of queens survive to the adult stage. The correct formation and persistence of polyfusomes has been suggested as a critical factor for ovariole survival. We have analyzed the developmental dynamics of F-actin and alpha-spectrin in fusomes of queen and worker larvae, and in juvenile-hormone-treated worker larvae. Small fusomes containing actin and spectrin can be detected in the ovaries of fourth instar larvae in both castes. After molting to the fifth instar, the actin-spectrin association persists in the enlarged fusomes of queen ovarioles. In workers, actin dissociates from the fusomal and cortical alpha-spectrin. Coinciding with the appearance of apoptosis markers, large agglomerates of actin are detectable in worker ovarioles. Treatment of fourth-instar worker larvae with juvenile hormone rescues ovarioles from apoptosis and maintains the actin-spectrin association. Juvenile-hormone-dependent actin-spectrin interaction is thus one of the earliest steps in the differentiation of a polymorphic ovary. Plasticity in ovariole numbers as a result of hormone-dependent fusome formation may be a more widespread phenomenon in insects, extending beyond caste polymorphism in highly eusocial Hymenoptera.