A potential diagnostic reagent for bovine cysticercosis

A fraction of larval Taenia hydatigena cyst fluid was shown to have high sensitivity and specificity in the enzyme-linked immunosorbent assay (ELISA) for the detection of bovine antibodies to the heterologous parasite Taenia saginata. This antigenically active lipoprotein fraction was isolated by ultracentrifugal density flotation using either ammonium sulfate (specific gravity = 1.231 g per ml) or NaCl/KBr (specific gravity = 1.225 g per ml), followed by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that this fraction was composed of high molecular weight (65,000 to 77,000 Mr) and low molecular weight (9,500 to 16,000 Mr) proteins. Electrophoresis under non-denaturing conditions in either acrylamide (5%) or agarose (1%) resulted in 1 major diffuse band staining for both protein and lipid. The high and low molecular weight proteins observed on SDS-PAGE under reducing conditions could not be resolved by gel filtration chromatography and emerged as a single lipoprotein peak. This T. hydatigena cyst fluid fraction appears promising as a diagnostic reagent in the ELISA for bovine cysticercosis.     

Cation transport in Escherichia coli. IX. Regulation of K transport

Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B. F.B. Walters, and W. Epstein. 1976. J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kpd and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high.     

The C-biogeochemistry of a Midwestern USA agricultural impoundment in context: Lake Decatur in the intensively managed landscape critical zone observatory

The damming of rivers has created hotspots for organic carbon sequestration and methane production on a global scale as the reservoirs intercept fluvial suspended and dissolved loads. To better understand how the C-biogeochemistry of a reservoir responds to watershed processes and evolves over time, Lake Decatur, located in the Intensively Managed Landscape Critical Zone Observatory (IML-CZO) was studied. Solid phase analyses (% organic C, C/N, δ¹³C, δ¹⁵N) of soils and sediments sampled from stream bank exposures, river suspensions, and the lake bottom were conducted to characterize organic C (OC) sources throughout the sedimentary system. Agriculturally-driven soil erosion rapidly altered lake bathymetry causing an evolution of sedimentary and OC deposition patterns, which in turn shaped where and when methane production occurred. A positive correlation between OC accumulation rate and porewater dissolved inorganic C (DIC) δ¹³C profiles indicates that methane generation is strongly influenced by OC burial rate. The sources of the lake bed particulate organic C (POC) have also evolved over time. Drowned vegetation and/or shoreline inputs were dominant initially in areas adjacent to the original river channel but were rapidly overwhelmed by the deposition of sediments derived from eroded agricultural soils. Eutrophication of the lake followed with the onset of heavy fertilizer application post-1960. This succession of sources is expected to be commonplace for reservoirs greater than?~?50–60 years old in agricultural settings because of the relative timing of tillage and fertilizer practices. The ¹³C/¹²C ratios of methane from Lake Decatur were more depleted in ¹³C than what is commonly expected for freshwater sedimentary environments. The ¹³C-depletion suggests that CO₂-reduction is the dominant methanogenic pathway rather than the anticipated acetate dissimilation process. The isotopic observations reveal that commonly held assumptions about methane production and its C-isotopic signature in freshwater systems are over-simplified and not strictly applicable to this system.     

Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma.

Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA. Specific proteolysis of protein synthesis initiation factor eIF-4 gamma occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs. Cleavage of eIF-4 gamma in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.e., foot-and-mouth disease viruses), the leader proteinase is responsible for this reaction. We describe here the purification to homogeneity of the Lb form of the leader proteinase expressed in Escherichia coli. The primary cleavage products of eIF-4 gamma obtained in vitro with purified leader or 2A proteinase are electrophoretically indistinguishable from those found during infection in vivo. However, additional proteolysis products of eIF-4 gamma are observed with the leader proteinase and the human rhinovirus type 2 2A proteinase in vitro. The cleavage site of the leader proteinase in eIF-4 gamma from rabbit reticulocyte was determined by sequencing the purified C-terminal cleavage product by automated Edman degradation. The cleavage site is between Gly-479 and Arg-480 and thus differs from that of rhinovirus and enterovirus 2A proteinases, which cleave between Arg-486 and Gly-487.     

PCR-mediated chemical mutagenesis of cloned duplex DNAs

We describe an efficient, PCR-mediated protocol for random chemical mutagenesis of cloned duplex DNAs. The method involves a single molecular cloning step and is compatible with a wide variety of recombinant DNA vectors. To illustrate the procedure, we report the nitrous acid mutagenesis of a human ribosomal protein S14 cDNA fragment.