Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.     

Combined Oxygen and Nitrous Oxide Microsensor for Denitrification Studies

The construction of a microsensor which can be used to measure O₂ and N₂O simultaneously is described. The microsensor exhibited a linear response to both O₂ and N₂O, and the response to N₂O was independent of the O₂ concentration and vice versa. The N₂O detection limit of a microsensor with a tip diameter of 20 μm was around 1 μmol liter⁻¹. The signals for O₂ and N₂O were affected by hydrogen sulfide, but other interfering agents were not observed in the biofilms and sediments analyzed. Microprofiles of O₂ and N₂O were measured in a biofilm which was exposed to acetylene to block the N₂O reductase activity of denitrifying bacteria. O₂ penetrated about 0.5 mm into the biofilm and was not affected by acetylene, but the N₂O concentration at 1.4 mm depth increased from 32 to 411 μmol liter⁻¹ after the addition of the inhibitor. The shape of the N₂O profile after the addition of acetylene showed that denitrification (denitrifying activity) was detectable in all anoxic layers of the biofilm.     

Proposed virulence factors among coagulase-negative staphylococci isolated from two healthy populations

Proposed virulence factors, including multiple antibiotic resistance and slime-mediated adherence, among coagulase-negative staphylococci colonizing healthy individuals from two different study populations were examined. Resistance to methicillin was more commonly seen than initially anticipated. In addition, adherence characteristics, as quantitated by a microtiter plate method, were very similar to those of strains of coagulase-negative staphylococci causing nosocomial infections.     

Pollen as a chronometer and sediment tracer,Burrinjuck Reservoir,Australia

Pollen analysis is widely used to reconstruct vegetation and land use histories, but can also provide sedimentological information. At Burrinjuck Reservoir, in south-eastern Australia, annual grass pollen peaks are used to distinguish each year's sediment, even when there are no visible laminations. In conjunction with other dating methods, this allows the determination of year by year influxes of all sediment components. Pollen grains in the Burrinjuck sediments are shown to be predominantly waterborne so that they can be used to trace sediment to its source in particular vegetation stands. Pollen concentration and the proportion of damaged pollen might also distinguish sediment eroded from topsoils and that from subsoils. Pollen analysis can thus be used to locate specific erosion events in both time and space.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Assessment of tibial stiffness by vibration testing in situ--II. Influence of soft tissues, joints and fibula

The influence of soft tissues and joints on the vibration of the human tibia was examined by modal analysis on amputated lower limbs, where the soft tissues and the fibula were dissected gradually. Measurements were made in two different set ups, IFR and BRA, which were both designed to monitor fracture healing. In IFR, vibrations are generated by hammer impact on a relaxed hanging lower leg, with the knee flexed. Resonant frequencies are determined by a computer Fourier transform procedure. In BRA, a steady state vibration is induced in a lower leg, supported near the ankle and the tibial tuberosity, using an electromagnetic shaker. Resonant frequencies are determined from the maxima in vibration amplitudes. In both set ups the soft tissues have a similar influence on the vibration of the tibia: the skin hardly influences the determined modal parameter. The mass of the muscles influences both the resonant frequency and the damping. The fibula has a stiffening effect on the tibia. The influence of the joints is small in the IFR-set up: the tibia vibrates in conditions close to those for the free-free vibration. In the BRA-set up, the supports determine the boundary conditions.     

Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal?

The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. trans-Stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. In addition, we find cystine exodus from the lysosomal fraction of cystinotic human fibroblasts to be greatly retarded as compared to that of normal human fibroblasts with half-times of exodus similar to those reported for the lysosomes of cystinotic and normal human leukocytes (Gahl, W. A., Tietze, F., Bashan, N., Steinherz, R., and Schulman, J. D. (1982) J. Biol. Chem. 257, 9570-9575). In contrast, normal and cystinotic human fibroblasts did not show any differences with regard to lysine efflux or its trans-stimulation by cationic amino acids. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.     

Defense reactions of mosquitoes to filarial worms: potential mechanism for avoidance of the response by Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and the black-eyed Liverpool (LVP) and Rocke-feller (RKF) strains of Aedes aegypti against intrathoracically inoculated Brugia pahangi microfilariae (mff) that previously had penetrated LVP, RKF, or A. trivittatus midguts in vitro was assessed at 1, 3, and 5 days postinoculation (PI). LVP and RKF midgut-derived mff almost totally avoided the melanization response and developed normally in LVP strain A. aegypti, and although over 90% of these mff died by 5 days PI in RKF mosquitoes, the majority of these were not melanized. A. aegypti midgut-derived mff also were able to avoid the response of A. trivittatus in 33–43% of the cases. Penetration through A. trivittatus midguts, however, did not significantly affect the ability of mff to avoid the melanization response in any of the mosquitoes examined. Allogeneic and xenogeneic tissue inplants were accepted by all three mosquito species examined. Data presented support the hypothesis that mff avoid immune recognition in compatible mosquitoes by coating themselves with midgut material(s) during penetration of the midgut in their migration to the hemocoel.     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

New mutations affecting induced mutagenesis in yeast

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4–38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4⁺ and REV5⁺ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.