ABSTRACT
Chitosan microspheres were prepared by ionic gelation process with sodium sulfate for nasal vaccine delivery. Bordetella Bronchiseptica Dermonecrotoxin (BBD) as a major virulence factor of a causative agent of atrophic rhinitis (AR) was loaded to the chitosan microspheres for vaccination. Morphology of BBD-loaded chitosan microspheres was observed as spherical shapes. The average particle sizes of the BBD-loaded chitosan microspheres were about 2.69 microm. More BBD was released with an increase of molecular weight of chitosan and with an increase of medium pH in vitro due to weaker intermolecular interaction between chitosan and BBD. Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO) from RAW264.7 cells stimulated with BBD-loaded chitosan microspheres were gradually secreted, suggesting that released BBD from chitosan microspheres had immune stimulating activity of AR vaccine.
Subject(s)
Bacterial Toxins/pharmacokinetics , Bacterial Vaccines/pharmacokinetics , Bordetella bronchiseptica/metabolism , Chitin/analogs & derivatives , Chitin/pharmacokinetics , Microspheres , Transglutaminases/pharmacokinetics , Virulence Factors, Bordetella/pharmacokinetics , Animals , Bordetella bronchiseptica/drug effects , Cell Line , Chitin/ultrastructure , Chitosan , Delayed-Action Preparations/pharmacokinetics , Light , Mice , Microscopy, Electron, Scanning , Scattering, Radiation , Swine , Transglutaminases/ultrastructureABSTRACT
Heterotrimeric (alphabetagamma) G proteins interact with sensory receptors to transduce signals to downstream effectors in eukaryotes. We previously reported that GNA-1 from Neurospora crassa is a microbial member of the Galphai family found in higher organisms. Deletion of gna-1 leads to female sterility, slower growth rates on normal and hyperosmotic solid medium, and increased resistance to heat and oxidative stress. In this study we compare mammalian genes for proteins of the Galphai sub-family (Galphai, Galphao, Galphat and Galphaz), and Galphas (which is not a member of the Galphai family) with the N. crassa gna-1 gene with respect to their ability to complement deltagna-1 phenotypes. Northern analysis detected full-length transcripts of all these genes, except that for Galphai, in N. crassa transformants. Measurements of pertussis toxin-catalyzed ADP-ribosylation and Western analysis showed that the GNA-1, Galphaz, Galphao and Galphas proteins were present in the respective transformed strains. Strains in which the mammalian Galpha protein could be detected were subjected to phenotypic testing. During the vegetative cycle, none of the mammalian Galpha genes complemented the thermotolerance phenotype of deltagna-1. However, the three expressed mammalian Galpha genes achieved at least partial complementation of the defects in vegetative apical extension rate. cAMP levels did not correlate with restoration of vegetative growth rate by the mammalian genes. During the sexual cycle, Galphao was the only mammalian Galpha gene that rescued the defect in female fertility characteristic of deltagna-1 strains. Alignment of GNA-1, Galphaz, Galphao and Galphas protein sequences revealed correlations between the observed complementation pattern and the degree of identity to GNA-1 in various functional motifs. The finding that Galphac gave the best restoration of vegetative growth but could not restore normal female fertility implies that GNA-1 regulates different pathways that are important for vegetative and sexual growth in N. crassa.
Subject(s)
Fungal Proteins , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Genetic Complementation Test , Heterotrimeric GTP-Binding Proteins/genetics , Neurospora crassa/genetics , Amino Acid Sequence , Animals , Cattle , Cellophane , Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Gene Deletion , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Mammals , Mice , Molecular Sequence Data , Neurospora crassa/drug effects , Neurospora crassa/physiology , Pertussis Toxin , Phenotype , Potassium Chloride/pharmacology , Reproduction , Sequence Alignment , Sequence Homology, Amino Acid , Sodium Chloride/pharmacology , Sorbitol/pharmacology , Virulence Factors, Bordetella/pharmacokineticsABSTRACT
In vivo administration of pertussis toxin is often used to study the involvement of guanine nucleotide binding proteins in signal transduction. Especially when it is administered in the brain the effect is often poor. This could be due to the fact that pertussis toxin does not reach the area of interest. To evaluate the extent to which pertussis toxin is distributed in rat brain after intraventricular injection, different techniques were used. Immunohistochemical studies with an antibody against pertussis toxin showed that immunoreactivity was limited to periventricular brain structures less than 0.5 mm from the lumen. The highest immunoreactivity was seen 16-24 h after injection. After 96 h the labeling was very weak. The proportion of guanine nucleotide binding proteins that were ADP-ribosylated by in vivo injection of pertussis toxin into the ventricles as assessed by in vitro [32P]-back-ADP-ribosylation was very low 48 h after the injection, in all regions studied. Direct injection of pertussis toxin into the brain caused a marked ADP-ribosylation localized to the region injected that was maximal at 72 h after injection. At 96 h there were also effects after control injections, indicating non-specific effects. Synaptosomal membranes and other membranes were equally affected by pertussis toxin. The results suggest that in studies regarding the effect of pertussis toxin treatment on signal transduction, the toxin must be injected very close to the brain region of interest and, furthermore, that the rats should be killed 48-72 h after injection. In case of lack of effect on the response of interest one should examine whether the ADP-ribosylation of pertussis toxin-sensitive guanine nucleotide binding proteins in the area of concern has been affected.
