Maternal or fetal origin of rhesus monkey (Macaca mulatta) amniotic fluid leukocytes can be identified by polymerase chain reaction using the zinc finger Y gene.

Leukocytes can be found in substantial numbers within the intrauterine tissues and amniotic fluid of women, and play a central role in the pathophysiology of infection-related preterm labor by their production of proinflammatory mediators. It remains unclear whether these leukocytes represent a fetal immune response, a maternal response, or a combination of the two. The objective of this study was to develop a test in the rhesus monkey (Macaca mulatta) suitable for determining the percentage of male fetal cells present in a population of leukocytes recovered from blood or amniotic fluid. We found inadequate specificity for rhesus monkey cells using commercial human Y-chromosome paint kits (fluorescence in situ hybridization (FISH)). Human-specific primers for the repetitive Y chromosome DYZ-1 locus employed in the polymerase chain reaction (PCR) produced an unacceptable percentage of false positives. However, we successfully developed a PCR-based test using rhesus-specific primers for the zinc finger Y (ZFY) locus. Densitometry of PCR products from known ratios of male and female adult peripheral leukocytes generated a linear standard curve which provided quantitative results and required only 400 cells per sample. The rhesus beta globin (RBG) gene served as an internal control. The PCR test correctly discriminated the sex of peripheral leukocytes in 20 adult males, 20 adult females, two male fetuses, and one female fetus. Serial samples of amniotic fluid from four chronically catheterized rhesus monkeys bearing male fetuses were used to confirm the utility of this assay for quantifying fetal cells in amniotic fluid. In conclusion, we have developed a PCR test which is suitable for distinguishing male from female cells in adult and fetal blood and in amniotic fluid, which lends itself to a variety of diagnostic and biologic applications in the rhesus monkey and potentially in other nonhuman primates.     

Isolation of macrocyclic and non-macrocyclic trichothecenes (stachybotrys and fusarium toxins) from the Environment of 200 III Sport Horses

Satratoxins H and G, verrucarin J, and roridin E were isolated from the bedding straw of 200 sport horses exhibiting typical symptoms of stachybotryo-toxicosis. At the same time, the oat feed consumed by the horses contained non-macrocyclicFusarium trichothecenes: T-2 toxin and diacetoxyscirpenol.     

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.     

Influence of soil variables on in situ plasmid transfer from Escherichia coli to Rhizobium fredii.

A model system was established to determine whether intergeneric plasmid transfer occurs in soil and how various soil variables affect the rate of plasmid transfer. The donor bacterium, Escherichia coli HB101 carrying plasmid pBLK1-2 (pRK2073::Tn5), and the recipient bacterium, Rhizobium fredii USDA 201, were inoculated into a sterile Adelphia fine-sandy-loam soil. Transconjugants were enumerated by direct plating on antibiotic-amended HM [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 2-(N-morpholino) ethanesulfonic acid] salts medium. Randomly chosen transconjugants were verified by serological typing and Southern hybridization with a Tn5 gene probe. The maximum transfer frequency was observed after 5 days of incubation (1.8 x 10(-4) per recipient). The influences of clay (0 to 50% addition), organic matter (0 to 15% addition), soil pH (4.3 to 7.25), soil moisture (2 to 40%), and soil incubation temperature (5 to 40 degrees C) on plasmid transfer were examined. Maximum transfer frequencies were noted at a clay addition of 15%, an organic matter addition of 5%, a soil pH of 7.25, a soil moisture content of 8%, and a soil incubation temperature of 28 degrees C. These results indicate that intergeneric plasmid transfer may occur in soil and that soil variables may significantly affect the rate of transfer.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Covalent immobilization of avidin on glassy carbon electrodes as the basis for multivalent biosensors.

One of the most crucial steps for the successful construction of a biosensor is the appropriate and reproducible coupling of the biological part (e.g. enzyme, antibody) to the inorganic moiety of the device (e.g. electrode, microchip). In this paper three methods of immobilization of avidin to a glassy carbon electrode are described. Depending on the type of immobilization, avidin may lose its biological activity as determined by an enzyme immunoassay, using biotinylated reagents. If avidin is covalently bound to the glassy carbon electrode via the bridge molecule 4.4'-diaminodiphenylamine, the biological activity is retained. About 1.5 pmol of avidin can be bound to the electrode (3 mm in diameter), resulting in a nearly complete monolayer of protein.     

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.