Use of an Exotic Carbon Source To Selectively Increase Metabolic Activity and Growth of Pseudomonas putida in Soil

Respiration and growth of Pseudomonas putida PpG7, containing catabolic plasmid NAH7, was determined in three agricultural field soils amended with the carbon source salicylate. The addition of salicylate to soil significantly increased the population of PpG7. However, there was a lack of relationship between microbial numbers and activity as determined by evolution of CO₂. In soils containing 30 to 1,500 μg of salicylate per g, metabolic activities of PpG7 peaked between 18 and 42 h and population densities increased approximately 10¹-to 10⁵-fold. However, the metabolic activity of PpG7 rapidly declined after salicylate was utilized, whereas peak population densities were maintained for the duration of the experiments (5 to 7 days). Thus, elevated population densities of PpG7 were represented by inactive cells. Soil type had only minor effects on respiration rates or growth curves of PpG7 when amended with comparable concentrations of salicylate. Respiration and growth rates were optimal at concentrations between 300 and 1,000 μg of salicylate per g in the test soils. At 1,500 to 2,500 μg/g, respiration and growth of PpG7 were initially suppressed, but after a short lag time both attained levels similar to or greater than those resulting from the use of lower concentrations of salicylate. The culturing of PpG7 on a salicylate-amended medium to induce salicylate-degradative enzymes did not affect the lag time before utilization of salicylate in soil. Although PpG7 competed well with fungi for the substrate, suppression of fungal populations with cycloheximide resulted in significantly increased population densities of PpG7 in two of three soils amended with salicylate. The beneficial activities of bacteria in soil are discussed in relation to population density, population metabolic activity, and selective carbon source utilization.     

Numerical method for equilibrium fluorescence polarization

A computer program for making first approximations of equation constants, and for fitting the equilibrium fluorescence polarization equation to data by successively improving the constant values is described. The techniques used in making the first approximations represent a substantial improvement over methods previously available.     

Pulmonary surfactant synthesis. A highly active microsomal phosphatidate phosphohydrolase in the lung.

Lung cell-free homogenate, which contains about twice the units of phosphatidate phosphohydrolase per mg of protein compared to liver, was fractionated by differential centrifugation and the fractions were assayed for phosphatidate phosphohydrolase and marker enzymes of endoplasmic reticulum, mitochondria, and lysosomes. Over 60% of the lung phosphatidate phosphohydrolase was associated with the endoplasmic reticulum, compared to 50% of the total liver enzyme. Thus a major portion of the more active lung enzyme is potentially involved in lipid biosynthesis by the endoplasmic reticulum. Less than 0.2% of the total lung enzyme was found in a lamellar body fraction, consistent with previous findings. The lung microsomal phosphohydrolase was specific for lipid substrates, showing equal activity towards phosphatidic acid or lysophosphatidic acid and relatively low activities towards glycerophosphates. It had a neutral pH optimum, similar to the liver enzyme, but differed somewhat in its relative activity at extremes of pH. Stability at 65 degrees C was greater for the lung enzyme. Fluroide inhibited lung (or liver) microsomal phosphatidate phosphohydrolase, while tartrate, MgCl2, or EDTA had no effect. The presence of a high activity of phosphatidate phosphohydrolase in lung endoplasmic reticulum is consistent with the rapid synthesis of pulmonary surfactant phosphatidylcholine.     

Structure of adeno-associated virus type 4

Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-A resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.     

Beauveria bassiana affects immature stage development of Prostephanus truncatus (Coleoptera: bostrichidae) in stored maize

Recent research in Ghana has demonstrated the effectiveness of an isolate of B. bassiana, sensu lato (IMI 389521) from the U.K. against the larger grain borer Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae), a major pest of stored maize. To determine whether this isolate is effective on immature stages of P. truncatus, a laboratory study of the response of immature stages (egg, larvae and pupae) of P. truncatus in maize grains to two concentrations (1?×?10⁹ and 3.16?×?10⁹?cfu/kg maize) of B. bassiana, IMI 389521 formulated with Entostat? and kaolinite was undertaken. Adult emergence, per cent survival of adults that emerged and the number of larvae in each immature stage were assessed after 45 days. Apart from the egg experiment, higher numbers of adults emerged in grains containing larvae and pupae treated with B. bassiana product compared to the untreated maize control. This notwithstanding, survival of emerged adults of P. truncatus was greatly reduced in B. bassiana treatments (<10%) compared with 75–95% in the controls. Surface treatment with B. bassiana on pre-infested maize showed a significant effect on the developmental biology of P. truncatus in Ghana.     

Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.     

Dynamic surface properties of phosphatidylglycerol-dipalmitoylphosphatidylcholine mixed films

This paper studies the dynamic surface pressure-area (π-A) behavior of phosphatidylglycerol (PG) with a mixed fatty acid distribution (bacterial) in pure and binary mixed films with dipalmitoyl phosphatidylcholine (DPL). At 23°C, bacterial PG films generate maximum dynamic surface pressures of only 48–49 dyn/cm on a 0.15 M sodium chloride subphase for both dilute and surface excess initial conditions. By contrast, binary mixed films of 90:10 DPL/PG reach maximum π values of the order of 70 dyn/cm at similar conditions, the same as for pure DPL films. A collapse plateau ratio criterion is used to show that respreading after dynamic compression past film collapse is enhanced in 90:10 DPL/PG films as compared to pure DPL for a dilute surface initial condition, but not for the surface excess condition, at room temperature. Respreading in pure bacterial PG films is also slightly improved over corresponding pure DPL films for some initial conditions at 23°C, but the magnitude of this effect is not as large as might be expected from the significant unsaturated and cyclopropane fatty acid percentage present in bacterial PG. Differential Scanning Calorimetry (DSC) measurements on DPL/PG mixtures show decreasing T_c with peak broadening as the percentage of bacterial PG is increased. The experiments here do not establish a clearly required functional role for 10% PG in pulmonary surfactant surface behavior. Further surface studies are suggested before long-term clinical trials of PG containing mixtures for exogenous replacement therapy in Neonatal Respiratory Distress Syndrome (RDS) are initiated on a widespread basis.