Perspectives on measurement of denitrification in the field including recommended protocols for acetylene based methods

Of the biogeochemical processes, denitrification has perhaps been the most difficult to study in the field because of the inability to measure the product of the process. The last decade of research, however, has provided both acetylene and¹⁵N based methods as well as undisturbed soil core andin situ soil cover sampling approaches to implementing these methods. All of these methods, if used appropriately, give comparable results. Thus, we now have several methods, each with advantages for particular sites or objectives, that accurately measure denitrification in nature. Because of the general usefulness of the acetylene methods, updated protocols for the following three methods are given: gas-phase recirculation soil cores; static soil cores; and the denitrifying enzyme assay also known as the phase 1 assay. Despite the availability of these and other methods, denitrification budgets remain difficult to accurately establish in most environments because of the high spatial and temporal variability inherent in denitrification. Appropriate analysis of those data includes a distribution analysis of the data, and if highly skewed as is typically the case, the most accurate method to estimate the mean and the population variance is the UMVUE method (uniformly minimum variance unbiased estimator). Geostatistical methods have also been employed to improve spatial and temporal estimates of denitrification. These have occasionally been successful for spatial analysis but in the attempt described here for temporal analysis the approach was not useful.Discussions of the importance of denitrification have always focused on quantifying the process and whether particular measured quantities are judged to be a significant amount of nitrogen. A second line of evidence discussed here is the extant genetic record that results from natural selection. These analysis lead to the conclusion that strong selection for denitrification must currently be occurring, which implies that the process is of general significance in soils.     

Rearrangement,amplification, and assortment of mitochondrial DNA molecules in cultured cells of Brassica campestris

Summary We compared Brassica campestris mitochondrial and chloroplast DNAs from whole plants and from a 2-year-old cell culture. No differences were observed in the chloroplast DNAs (cpDNAs), whereas the culture mitochondrial DNA (mtDNA) was extensively altered. Hybridization analysis revealed that the alterations are due entirely to rearrangement. At least two inversions and one large duplication are found in the culture mtDNA. The duplication element is shown to have the usual properties of a plant mtDNA high frequency recombination repeat. The culture mtDNA exists as a complex heterogeneous population of rearranged and unrearranged molecules. Some of the culture-associated rearranged molecules are present in low levels in native plant tissue and appear to have sorted out and amplified in the culture. Other mtDNA rearrangements may have occurred de novo. In addition to alterations of the main mitochondrial genome, an 11.3 kb linear mtDNA plasmid present in whole plants is absent from the culture. Contrary to findings in cultured cells of other plants, small circular mtDNA molecules were not detected in the B. campestris cell culture.     

Cilia on bovine mammary epithelium: ultrastructural observations

Ultrastructural examination of bovine mammary tissues revealed the presence of 9+0 or primary cilia protruding from surfaces of alveolar epithelial and myoepithelial cells. Cilia of epithelial cells protruded approximately 1200 nm into lumina of alveoli and arose from a basal body centriole, the associated centriole of the diplosome, and an accessory rootlet system. Cilia on epithelial cells were more frequently observed than cilia on myoepithelial cells. Occasional cilia made contact with macrophages in the alveolar lumen. The structures were more commonly found in tissues from nonlactating cows, and most were observed in the ventral portion of the mammary gland.     

Measurement of the extent of electron transfer to the bacteriopheophytin in the M-subunit in reaction centers of Rhodopseudomonas viridis

We have measured the extent of flash-induced electron transfer from the bacteriochlorophyll dimer, P, to the bacteriopheophytin in the M-subunit, H_M, in reaction centers of Rhodopseudomonas viridis. This has been done by measuring the transient states produced by excitation of reaction centers trapped in the PH_L ^–H_M state at 90 K. Under these conditions the normal forward electron transfer to the bacteriopheophytin in the L-subunit, H_L, is blocked and the yield of transient P⁺H_M ^– can be estimated with respect to the lifetime of P^*. Under these conditions flash induced absorbance decreases of the bacteriochlorophyll dimer 990 nm band suggest that a transient P⁺ state is formed with a quantum yield of 0.09±0.06 compared to that formed during normal photochemistry. These transient measurements provide an upper limited on the yield of a transient P⁺ H_M ^– state. An estimate of 0.09 as the yield of the P⁺ H_M ^– state is consistent with all current observations. This estimate and the lifetime of P^* suggest that the electron transfer rate from P^* to H_M, k_M, is about 5 × 10⁹ sec^–1 (_M = 200ps). These measurements suggest that the a branching ratio k_L/k_M is on the order of 200. The large value of the branching ratio is remarkable in view of the structural symmetry of the reaction center. This measurement should be useful for electron transfer calculations based upon the reaction center structure.     

Construction of a shuttle vector for Zymomonas mobilis

Summary An Escherichia coli-Zymomonas mobilis shuttle vector was constructed from a 15.5 kb native plasmid of ZM6 00 and the E. coli plasmid, pBR329. Integrative transfer of this shuttle vector from E. coli to Z. mobilis was achieved with the aid of the mobilizing plasmid, pRK2013. The shuttle vector was stable in Z. mobilis for at least 300 generations without antibiotic selection.Offprint requests to: S. F. Delaney     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Interactions of amiloride and small monovalent cations with the epithelial sodium channel. Inferences about the nature of the channel pore.

The voltage dependence of amiloride-induced inhibition of current flow through apical membrane sodium channels in toad urinary bladder was studied at different ionic conditions. The "inert" salt N-methyl-D-glucamine HCl (NMDG HCl) affected neither the apparent inhibition constant (Kl) for the amiloride-induced current inhibition nor the apparent fraction of the transmembrane voltage that falls between the mucosal solution and the amiloride-binding site (delta). When NMDG+ was replaced with Na+, Kl increased, reflecting amiloride-Na+ competition, whereas delta was unchanged. Similar results were obtained with another permeant cation, Li+. When NMDG+ was replaced by K+, an impermeant but channel-blocking cation, Kl increased whereas delta decreased. Similar results were obtained using another impermeant, channel-blocking cation guanidinium. The results are interpreted on the premise that Na+ and K+ compete with amiloride by binding to cation binding sites within the channel lumen such that ion occupancy of these sites vary with voltage. Occupancy by K+, which cannot traverse the channel, will increase as the mucosal solution becomes positive, relative to the serosal solution. Occupancy by Na+, which can traverse the channel, is comparatively voltage independent. Ion movement through the channels was simulated using discrete-state kinetic models. Two types of models could describe the shape of the current-voltage relationship and the voltage dependence of the amiloride-induced channel block. One model had a single ion-binding site with a broad energy barrier at the inner (cytoplasmic) side of the site. The other model had two binding sites separated from each other and from the aqueous solutions by sharp energy barriers.     

Murine embryonal carcinoma cell-surface sialyl Le is present on a novel glycoprotein and on high-molecular-weight lactosaminoglycan

Carbohydrate-specific monoclonal antibodies were used to demonstrate the expression of a new membrane glycoprotein on F9 murine embryonal carcinoma cells. Sialyl Le^x was detected using monoclonal antibody FH6 in a sensitive, cell monolayer radioimmunoassay. The antigen codistributed in gel filtration of a crude homogenate and in a membrane-enriched fraction with two known lactosaminoglycan markers, i and SSEA-1 (Le^x or X hapten). Sialyl Le^x was further shown to be carried by a novel glycoprotein, termed small lactosaminoglycan-like glycoprotein (sLAG) which could be purified by immunoaffinity chromatography. In two-dimensional polyacrylamide gel electrophoresis this glycoprotein had an apparent molecular weight of 45 kDa and a pI of about 6.5. The more differentiated cell line PYS-2 also expressed sialyl Le^x and i antigens but not Le^x, and FH6-reactive sLAG could be extracted from PYS-2 membranes. Sialylation of fucosylated type 2 carbohydrate chains (X haptens) thus may be an early modification of embryonic carbohydrate antigens.     

Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of ¹⁸O- and ¹⁶O-labeled 3-methylcatechol from toluene in an atmosphere of ¹⁸O₂ and ¹⁶O₂ established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.     

Bacteriophage Transport in Sandy Soil and Fractured Tuff

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to 40% of the void volume but did not adsorb. Dispersion in the field was similiar to that observed in the laboratory. The phage f2 was largely excluded from the porous matrix of the two fractured-rock cores studied, coming through 1.2 and 2.0 times later than predicted on the basis of fracture flow alone. Because of matrix diffusion, nonsorbing solutes were retarded by over a factor of three relative to fracture flow. The time for a solute tracer to equilibrate with the porous matrix of 6.5-cm-diameter by 25-cm-long cores was measured in days. Results of both granular-medium and fractured-rock experiments illustrate the inability of a solute tracer to provide estimates for dispersion and effective porosity that are applicable to a colloid. Bacteriophage can be used to better estimate the maximum subsurface transport rate of colloidal contaminants through a porous formation.