Intranasal immunization using the B subunit of the Escherichia coli heat-labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen

The plasmid pBRD026, which directs expression of the B subunit of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3' end of the gene. An oligonucleotide linker containing restriction sites for BglII and SpeI was inserted at the SpeI site at the 3' end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly-Pro-Gly-Pro which we propose acts as a 'hinge' between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetella pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice.     

Extracellular calcium mimics the actions of platelet-derived growth factor on mouse fibroblasts.

Microprecipitates of calcium phosphate (CaPO4) can substitute for platelet-derived growth factor (PDGF) to stimulate the growth of cultured 3T3 cells. In two-part complementation assays, CaPO4 behaves as a PDGF-like "competence factor"--that is, the mitogenic response to CaPO4 is enhanced synergistically by "progression factors" contained in platelet-poor plasma. In studies described here, we show that early cytoplasmic and intranuclear events in the mitogenic response to CaPO4 are equivalent to those induced by PDGF. However, no net increase in tyrosine kinase activity of either the PDGF-alpha or PDGF-beta receptor is seen following exposure to CaPO4. Our data suggest that calcium acts within the cell, regulating events which normally proceed from activation of PDGF receptors. Alternatively, microprecipitates of CaPO4 could act externally by activating a growth factor receptor which escapes detection with available reagents.     

TWO NEUTRALITY TESTS OF Y-LINKED RDNA VARIATION IN DROSOPHILA MELANOGASTER

The evolutionary significance of Y-chromosomal ribosomal DNA sequence variation was tested by two different means. A single sample of males and females was collected from a peach orchard in central Pennsylvania. Wild-caught males and sons of wild-caught, wild-inseminated females were crossed to virgin females having an X-linked rDNA deficiency. Genomic DNA from male progeny of these crosses was extracted and digested with the single restriction endonuclease, DraI. Southern blots of these digestions, when probed with the complete rDNA probe, revealed 10 distinct patterns of restriction fragments. A chisquare test for the homogeneity of the frequency distributions of the sample of wild males and sons of wild females failed to reject a neutral null hypothesis. The allele frequency configuration was tested with the Ewens-Watterson test, and the departure from the infinite alleles neutral model was not significant. Simulations were performed to test the sensitivity of the tests to misclassification and to quantify the power of the two tests.     

Effects of planktivore abundance on chlorophyll-a and Secchi depth

We used two analyses to test the hypothesis that planktivore abundances contribute to the residual variations of Secchi depth or chlorophyll-a plotted with respect to mean summer epilimnetic total phosphorus. The first analysis involved 15 lake years of data from six lakes. The data set comprised mark-recapture assessments of piscivore and planktivore numbers and estimates of mean summer chlorophyll-a, total phosphorus and Secchi depth. We found that residual chlorophyll-a variation was not significantly (p>0.05) correlated with planktivore densities, but that planktivore densities did contribute (p<0.02) to the residual variation of Secchi depth on mean total phosphorus. The second analysis included all of the data used in the first plus an additional 13 lake years of data from the literature. These data showed that the percentage of the total fish community comprising planktivores did not significantly (p>0.05) contribute to the residual variation in chlorophyll-a with respect to mean summer total phosphorus. Together, our results suggest that planktivore abundance has a significant cascading impact on water clarity, but no long term statistically significant impact on mean summer chlorophyll-a concentration.     

Stage-specific cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi

In this study we have shown that NHS-biotin and I¹²⁵-streptavidin can detect cuticular polypeptides of Ostertagia spp. The labelled polypeptide profile of intact nematodes is simple compared to the profile obtained by labelling homogenates. None of the major internal polypeptides are labelled and the subset of proteins labelled in intact nematodes appears to be mainly surface associated. The results presented here demonstrate that NHS-biotin may be used as a reagent for the analysis of surface polypeptides. The surface polypeptide profiles of the five major developmental stages (L1, L2, L3, L4 and adult) of Ostertagia circumcincta show a series of stage-specific molecules with no polypeptides common to all stages, indicating that the cuticle is a dynamic structure which changes throughout the life cycle. Similarity comparison of Ostertagia ostertagi L3 and L4 stage surface profiles showed that each stage is clearly distinct; comparison of these stages between the two species shows an overall similarity.     

Application of automated image analysis to demonstrate the correlation between ras p21 expression and severity of gliomas

We found a direct correlation between increasing ras p21 protein immunopositivity and severity of human glioma using computer-assisted, digital-image processing to quantify the amount of p21 immunoreactive to the monoclonal antibody RAP-5. We determined that there was a significant difference in reactivity between glioblastoma multiformes and more-differentiated astrocytomas (experiment-wise error less than 0.05). This result confirmed the conclusions made on the same tumors using standard light microscopy and visual examination. Immunohistochemistry quantized by automated image analysis may be a useful adjunct to current histopathological strategies since it decreases assay subjectivity and variation.     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)