Modified Agglutination Test for Pasteurella tularensis

Several modifications in technique were incorporated into the standard agglutination test for Pasteurella tularensis. Reciprocal shaking of all tubes in a Kahn shaker was introduced to increase the rate of agglutination and quantity of agglutinated cell mass, making it possible to report preliminary results within 4 hr. Increased incubation time at a higher temperature was used to favor the rate of agglutination. A serum control for each serum tested was necessary to detect false positive tests. Finally, a verification procedure with 5% NaCl used as the diluent was instituted to prevent these false positive reactions.     

A method for the estimation of 6-oxygenated metabolites of progesterone in urine

1. A method is described for the estimation of the 6-oxygenated metabolites of progesterone in urine. After hydrolysis the extract of urine is chromatographed on alumina to obtain a fraction containing mainly the 6-oxygenated metabolites. This fraction is oxidized to convert the metabolites into pregnane-3,6,20-triones, which are estimated as the dinitrophenylhydrazones. 2. The reliability criteria of the method are presented. Normal subjects excrete 0.1-0.6mg./day, and at the end of pregnancy values of 3.3-11.6mg./day are obtained.     

The plasma membrane of myxosporidian valve cells: freeze fracture data

Freeze fracturing of Myxosporidian spores reveals the occurrence of a continuous layer of transmembrane particles all over the surface area of the valve cells which form the spore envelope. These particles are densely packed all over the P face membrane. Due to their polygonal outline, their diameter (6-7 nm) and their central core, they resemble the particles forming the connections of gap junctions which metabolically couple the neighboring cells in animal tissues. In the present report, the role of the transmembrane particles is still hypothetical. However, they might represent a membrane structural specialization of the spores which are submitted to osmotic variations of the fluid external medium. Furthermore similar transmembrane particles are observed at the level of the septate junction which seals the valve cells. In this occurrence, they are arranged in a series of 40 double rows parallel to the suture of the spore envelope. These findings support the view that Myxosporidia are Metazoa and raise the problem of their origin.     

Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions

We have constructed, in the yeast Saccharomyces cerevisiae, a mosaic assembly of genes by in vivo recombination of partially homologous sequences. The approach was tested on cDNAs encoding functionally distinct mammalian cytochromes P-450 (P-450). The selection for recombinant cDNAs used the transformation of yeast cells, which required the recircularization of a linearized plasmid by recombination of two partially homologous cDNAs. Libraries of mosaic genes with bipartite or tripartite structures were generated by intramolecular and intermolecular recombination events. The presence of yeast promoter and terminator sequences on the flanking sides of the recombined cDNAs has allowed the synthesis of encoded mosaic proteins. A library of yeast clones producing recombinant mouse P-450 P1 and rabbit P-450 LM4 was screened using functional criteria to identify chimeras with shuffled substrate specificity. Restriction mapping of mosaic genes, biochemical analysis of the synthesized proteins, comparison of chimeric enzymes, and the alignment of sequences with bacterial P-450 camphor hydroxylase of known three-dimensional structure, all suggest that the P-450 P1 amino acid residues 203-238 play a major role in the control of cytochrome activity toward carcinogenic polycyclic aromatic hydrocarbons. Similar approaches to structure-function analysis are believed to be applicable to other protein families.     

Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes.

Dermaseptin, a 34 amino-acid residue antimicrobial polypeptide [Mor, A., Nguyen, V. H., Delfour, A., Migliore-Samour, D., & Nicolas, P. (1991) Biochemistry 30, 8824-8830] was synthesized and selectively labeled at its N-terminal amino acid with either 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), rhodamine, or fluorescein. The fluorescent emission spectra of the NBD-labeled dermaseptin displayed a blue-shift upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. Titrations of solutions containing NBD-labeled dermaseptin with SUV composed of zwitterionic or acidic phospholipids were used to generate binding isotherms, from which were derived surface partition constants of (0.66 +/- 0.06) x 10(4) M-1 and (2.8 +/- 0.3) x 10(4) M-1, respectively. The shape of the binding isotherms, as well as fluorescence energy transfer measurements, suggests that some aggregation of membrane-bound peptide monomers occurs in acidic but not in zwitterionic vesicles. The preferential susceptibility of the peptide to proteolysis when bound to zwitterionic but not to acidic SUV suggests that these aggregates might then penetrate a relatively short distance into the hydrophobic region of the acidic membrane. Furthermore, the results provide good correlation between the peptide's strong binding and its ability to permeate membranes composed of acidic phospholipids, as revealed by a dissipation of diffusion potential and a release of entrapped calcein from SUV.     

Transcobalamin II--cobalamin binding sites are present on rabbit germ cells.

Specific binding sites for rabbit transcobalamin II have been found on isolated adult rabbit germ cells. Scatchard analysis revealed a single class of binding sites for [57Co]cyanocobalamin-transcobalamin II with an association constant (Ka) of 1.3 x 10(10) M-1 and 700 sites per cell. Binding was reversible, saturable and calcium dependent. Electron microscope radioautography following incubation with iodinated transcobalamin II at 4 degrees C led to a detectable labeling mainly restricted to the plasma membrane.     

Endo.SK1: an inducible site-specific endonuclease from yeast mitochondria

Site-specific endonucleases have been found in various eukaryotic organelles such as mitochondria, chloroplasts and nuclei. These endonucleases initiate site-specific or homologous gene conversion in mitochondrial and nuclear DNA. Here, we report a new site-specific endonuclease activity, Endo.SK1, identified in mitochondria of strain SK1, a homothallic diploid strain ofSaccharomyces cerevisiae. Nucleotide sequences around the Endo.SK1-cleavage sites are different from those of known yeast site-specific endonucleases. The Endo.SK1 activity is, at least partly, specified by a gene in the SK1-derived mitochondria. A novel feature of the Endo.SK1 activity is its inducibility: the endonuclease activity was induced by ca. 40-fold by transfer of cells from a glucose medium into an acetate medium, and was then repressed. This transient induction was independent of the ploidy level of the cells, and coincided with induction of fumarase, a mitochondrial enzyme involved in the TCA cycle. Co-induction and co-repression of the mitochondrial site-specific endonuclease activity and a respiration-related enzyme indicate that the endonuclease activity is regulated in response to physiological conditions, and suggest a possible role for the endonuclease in mitochondrial DNA metabolism.     

A proenkephalin A-derived peptide analogous to bovine adrenal peptide E from frog brain: Purification, synthesis, and behavioral effects

A peptide derived from the posttranslational processing of proenkephalin A was isolated from an extract of the brain of the European green frog Rana ridibunda and its primary structure established as: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg¹⁰-Pro-Glu-Trp-Trp-Gln-Asp-Tyr-Gln-Lys-Arg²⁰-Tyr-Gly-Gly-Phe-Met. The structure was confirmed by chemical synthesis. The peptide represents an amphibian equivalent of bovine adrenal peptide E [preproenkephalin A (206–230)-peptide] but the sequence contains two amino acid substitutions (Met¹⁵ → Gln and Leu²⁵ → Met) compared with the mammalian peptide. The data support previous hypotheses that the Leu-enkephalin sequence is not present in preproenkephalin A of amphibians. Intracerebroventricular injections of frog peptide E (10 and 100 ng) in mice had no significant effect on horizontal locomotor activity. The peptide, in doses up to 1 μg, had no effect on latency of escape jumping in the hot plate test and the peptide (100 ng) did not modify responses (paw licking, rearing, and escape jumping) in morphine-treated mice.     

Characterization of the Stable Maintenance of theShigella flexneriPlasmid pHS-2

pHS-2 is a 3-kb plasmid originally isolated fromShigella flexneriinfections associated with reactive arthritis in humans. This plasmid is stably maintained in many clinical isolates ofShigella flexneri.The nucleotide sequence of this plasmid displays two closely linked regions that may play a role in the maintenance of this plasmid. One region consists of a 250-bp locus showing a significant homology to the ColE1cersite. The results indicate that thecer-like site of pHS-2, like the ColE1cersite, acts as arecA-independent, site-specific recombination site involved in the resolution of multimers, requiring the presence of the host-encoded factors ArgR, PepA, XerC, and XerD. The second region consists of a 36-kDa open reading frame involved in generating resistance to the bactericidal effect of complement, which confers a selective advantage to cells containing this sequence. The results also indicate that pHS-2 can replicate in another species of Enterobacteriaceae (Escherichia coli) and is mobilized by the F plasmid.