Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.     

Functional significance of variation in bryophyte canopy structure

In most bryophytes, the thickness of boundary layers (i.e., unstirred layers) that surrounds plant surfaces governs rates of water loss. Architectural features of canopies that influence boundary layer thickness affect the water balance of bryophytes. Using field samples (9.3 cm diameter cushions) from 12 populations (11 species) of mosses and liverworts, we evaluated the relationship between canopy structure and boundary layer properties. Canopy structure was characterized using a contact surface probe to measure canopy depth along perpendicular transects at spatial scales ranging from 0.8 to 30 mm on 186 points per sample. Semivariance in depth measurements at different spatial scales was used to estimate three architectural properties: surface roughness (L(r)), the scale of roughness elements (S(r)), and fine-scale surface texture, the latter characterized by the fractal dimension (D) of the canopy profile. Boundary layer properties were assessed by evaporation of ethanol from samples in a wind-tunnel at wind speeds from 0.6 to 4.2 m/s and applied to characterize mass transfer using principles of dynamic similarity (i.e., using dimensionless representations of conductance and flow). In addition, particle image velocimetry (PIV) was used to visualize and quantify flow over two species. All cushions exhibited the characteristics of turbulent as opposed to laminar boundary layers, and conductance increased with surface roughness. Bryophyte canopies with higher L(r) had greater conductances at all wind speeds. Particle image velocimetry analysis verified that roughness elements interacted with flow and caused turbulent eddies to enter canopies, enhancing evaporation. All three morphological features were significantly associated with evaporation. When L(r), S(r), and D were incorporated with a flow parameter into a conductance model using multiple linear regression, the model accounted for 91% of the variation in mass transfer.     

Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells

Cytotoxic T lymphocytes (CTL) generated in C57BL/6 (H-2b) mice in response to infection with the serologically distinct herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were cross-reactive against target cells infected with either serotype. However, HSV-2-infected cells were shown to be much less susceptible to CTL-mediated lysis, and analysis through the use of HSV-1 X HSV-2 intertypic recombinants mapped the reduced susceptibility to a region contained within 0.82 to 1.00 map units of the HSV-2 genome. The study reported here was undertaken to determine the possible reasons for the reduced susceptibility of HSV-2-infected cells to lysis by CTL. Competition for the specific lysis of labeled HSV-1-infected cells by either HSV-1- or HSV-2-infected, unlabeled inhibitor cells and frequency analysis of the CTL precursor able to recognize HSV-1- and HSV-2-infected cells suggested that the reduced susceptibility of HSV-2-infected cells to lysis could be explained, at least in part, by reduced levels of target cell recognition. A determination of the surface expression of the critical elements involved in target cell recognition by CTL following infection with HSV-1 or HSV-2 revealed that all the major HSV-specific glycoprotein species were expressed. Infection with both HSV-1 and HSV-2 caused a reduction in the expression of the class I H-2 antigens. However, this reduction was much greater following infection with HSV-2. This suggested that one important factor contributing to reduced lysis of HSV-2-infected cells may be the altered or reduced expression of the class I H-2 self-antigens.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Formation of native hepatitis C virus glycoprotein complexes.

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.     

Nitrogen fixation in grassland and associated cultivated ecosystems

Summary Nitrogen fixation in the natural, Agropyron-Koeleria grassland ecosystem was studied using the C₂H₂-C₂H₄ and N¹⁵ assays. Small soil samples and also undisturbed soil cores were used for analyses. Both techniques indicated that grassland and associated cultivated soils had low fixation rates (0.6–1.8 kg/ha per 28 days in the laboratory and, 1 kg/ha per season under actual field conditions). Algal colonies (Nostoc spp.) on the soil surface were active fixers when the surface of the grassland was moist. However, their small biomass limits the extent of fixation in most areas. In native grassland, 16 legumes bore nodules. The three most common speciesVicia americana, Thermopsis rhombifolia andOxytropis sericea, all of which had active nodules, contributed 10 per cent of the total nitrogenase activity. The non-legumesElaeagnus commutata andShepherdia argentea were profusely nodulated with active nodules, but were confined to specific habitats. No nodules were found onArtemisia orOpuntia spp. The major, heterotrophic, asymbiotic bacteria in the soil were clostridia. These utilize substrates produced by aerobic cellulose and hemicellulose degrading organisms to fix N in anaerobic microsites. The C₂H₂:N₂ reduction ratio was 3 to 1 in large, aerobic core samples, but was greater under water-logged conditions where high fixation rates occurred.