Presence of the major progesterone-induced proteins of the sheep endometrium in fetal fluids

Allantoic and amniotic fluids were collected on Days 60 (n = 3), 100 (n = 4), and 140 (n = 3) of pregnancy. The presence of uterine milk proteins (UTM-proteins) in these samples was evaluated by Ouchterlony immunodiffusion and enzyme-linked immunoabsorbant assay (ELISA). Eight of ten samples of allantoic fluid and three of ten samples of amniotic fluid produced one or two immunoprecipitin bands against antiserum to UTM-proteins. Each band fused with immunoprecipitin bands from UTM-proteins purified from uterine fluid. Data from a semi-quantitative ELISA indicated that allantoic fluid from all ewes and amniotic fluid from six of ten ewes contained immunoreactive UTM-proteins. Concentrations of UTM-proteins in these fluids were not statistically affected by day of gestation (p greater than 0.10), but tended to decline as gestation advanced. Greater concentrations of UTM-proteins were detected in allantoic fluid than in amniotic fluid (p less than 0.05). The physical characteristics of the immunoreactive material in allantoic and amniotic fluids were examined by polyacrylamide gel electrophoresis and Western blotting. The immunoreactive material was found to possess pIs and molecular weights identical to UTM-proteins. These results indicate that fetal fluids contain material that reacts with antiserum to UTM-proteins and has physical properties similar to UTM-proteins. It is likely, therefore, that the UTM-proteins are transported across the placenta during gestation, perhaps to serve some function in the fetal compartment.     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

Cold-Induced Freezing Tolerance in Arabidopsis

Changes in the physiology of plant leaves are correlated with enhanced freezing tolerance and include accumulation of compatible solutes, changes in membrane composition and behavior, and altered gene expression. Some of these changes are required for enhanced freezing tolerance, whereas others are merely consequences of low temperature. In this study we demonstrated that a combination of cold and light is required for enhanced freezing tolerance in Arabidopsis leaves, and this combination is associated with the accumulation of soluble sugars and proline. Sugar accumulation was evident within 2 h after a shift to low temperature, which preceded measured changes in freezing tolerance. In contrast, significant freezing tolerance was attained before the accumulation of proline or major changes in the percentage of dry weight were detected. Many mRNAs also rapidly accumulated in response to low temperature. All of the cold-induced mRNAs that we examined accumulated at low temperature even in the absence of light, when there was no enhancement of freezing tolerance. Thus, the accumulation of these mRNAs is insufficient for cold-induced freezing tolerance.     

Below-ground herbivory promotes soil nutrient transfer and root growth in grassland

Extremely little is known about the ecosystem-level implications of below-ground herbivory, which often represents the dominant form of consumption of primary productivity. We provide the first empirical evidence that low levels of below-ground herbivory may promote soil nutrient flux and root growth of both host plants and companion plants. Low levels of white clover ( Trifolium repens L.) root infection by clover cyst nematodes ( Heterodera trifolii Goffart) increased root growth by 141% and 219% in the host plant and the uninfected neighbouring grass ( Lolium perenne L.), respectively. Root infection increased the size of the soil microbial biomass in the root zone and the transfer of ¹⁵N from the host plant to soil and the neighbouring grass. These data suggest that low amounts of below-ground herbivory may increase the transfer of plant carbon and nitrogen below-ground, leading to increases in root growth and soil nutrient recycling in grasslands. Presumably, such interactions will influence the competitive interactions between plant species, altering plant community structure in grasslands.     

Optimum activity of the phosphofructokinase from Ascaris suum requires more than one metal ion

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to give fructose 1,6-bisphosphate (FBP) using MgATP as the phosphoryl donor. As the concentration of Mg(2+) increases above the concentration needed to generate the MgATP chelate complex, a 15-fold increase in the initial rate was observed at low MgATP. The effect of Mg(2+) is limited to V/K(MgATP), and initial rate studies indicate an equilibrium-ordered addition of Mg(2+) before MgATP. Isotope partitioning of the dPFK:MgATP complex indicates a random addition of MgATP and F6P at low Mg(2+), with the rate of release of MgATP from the central E:MgATP:F6P complex 4-fold faster than the net rate constant for catalysis. This can be contrasted with the ordered addition of MgATP prior to F6P at high Mg(2+). The addition of fructose 2,6-bisphosphate (F26P(2)) has no effect on the mechanism at low Mg(2+), with the exception of a 4-fold increase in the affinity of the enzyme for F6P. At high Mg(2+), F26P(2) causes the kinetic mechanism to become random with respect to MgATP and F6P and with MgATP released from the central complex half as fast as the net rate constant for catalysis. The latter is in agreement with previous studies [Gibson, G. E., Harris, B. G., and Cook, P. F. (1996) Biochemistry 35, 5451-5457]. The overall effect of Mg(2+) is a decrease in the rate of release of MgATP from the E:MgATP:F6P complex, independent of the concentration of F26P(2).     

Functional rules for lac repressor�Coperator associations and implications for protein�CDNA interactions

The Lac repressor has been used as a tool to understand protein–DNA recognition for many years. Recent experiments have demonstrated the ability of the Lac repressor to control gene expression in various eukaryotic systems, making the quest for an arsenal of protein–DNA binding partners desirable for potential therapeutic applications. Here, we present the results of the most exhaustive screen of Lac repressor‐DNA binding partners to date, resulting in the elucidation of functional rules for Lac–DNA binding. Even within the confines of a single protein–DNA scaffold, modes of binding of different protein–DNA partners are sufficiently diverse so as to prevent elucidation of generalized rules for recognition for a single protein, much less an entire protein family.     

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

Background

Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.

Methods

In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.

Results

The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.

Conclusion

Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.     

Studies related to the synthesis of derivatives of 2,6-diamino-2,3,4,6-tetradeoxy-D-erythro-hexose (purpurosamine C), a component of gentamicin C12

Reduction of 1,6-anhydro-3,4-dideoxy-β-D-glycero-hex-3-enopyranos-2-ulose (levoglucosenone) with lithium aluminium hydride afforded principally 1,6-anhydro-3,4-dideoxy-β-D-threo-hex-3-enopyranose (3), which was converted into 3,4-dihydro-2(S)-hydroxymethyl-2H-pyran (8) following acid-catalysed methanolysis and reductive rearrangement of the resulting α-glycoside 4 with lithium aluminium hydride. 1,6-Anhydro-3,4-dideoxy-2-O-toluene-p-sulphonyl-β-D-threo-hexopyranose, prepared from 3, reacted slowly with sodium azide in hot dimethyl sulphoxide to give 1,6-anhydro-2-azido-2,3,4-trideoxy-β-D-erythro-hexopyranose, which was transformed into a mixture of methyl 2-acetamido-6-O-acetyl-2,3,4-trideoxy-α-D-erythro-hexopyranoside (10) and the corresponding β anomer following acid-catalysed methanolysis, catalytic reduction, and acetylation. Acid treatment of methyl 4,6-O-benzylidene-3-deoxy-α-D-erythro-hexopyranosid-2-ulose yielded the enone 15, which was readily transformed into methyl 6-O-acetyl-3,4-dideoxy-α-D-glycero-hexopyranosid-2-ulose (19). Procedures for the conversions of DL-8, 10, and 19 into methyl 2,6-diacetamido-2,3,4,6-tetradeoxy-α-D-erythro-hexopyranoside (methyl N,N′-di-acetyl-α-purpurosaminide C) have already been described.     

Genetic Variation in Fusarium Section Liseola from No-Till Maize in Argentina

Strains of Fusarium species belonging to section Liseola cause stalk and ear rot of maize and produce important mycotoxins, such as fumonisins. We isolated two species, Fusarium verticillioides (Gibberella fujikuroi mating population A) and Fusarium proliferatum (G. fujikuroi mating population D) from maize cultivated under no-till conditions at five locations in the Córdoba province of Argentina. We determined the effective population number for mating population A (N_e) and found that the N_e for mating type was 89% of the count (total population) and that the N_e for male or hermaphrodite status was 36%. Thus, the number of strains that can function as the female parent limits N_e, and sexual reproduction needs to occur only once every 54 to 220 asexual generations to maintain this level of sexual fertility. Our results indicate that the fungal populations isolated from no-till maize are similar to those recovered from maize managed with conventional tillage. We placed 36 strains from mating population A into 28 vegetative compatibility groups (VCGs). Of the 13 strains belonging to five multimember VCGs, only 2 isolates belonging to one VCG were clones based on amplified fragment length polymorphism (AFLP) fingerprints. Members of the other four multimember VCGs had an average similarity index of 0.89, and members of one VCG were no more closely related to other members of the same VCG than they were to other members of the population as a whole. This finding suggests that the common assumption that strains in the same VCG are either clonal or very closely related needs to be examined in more detail. The variability observed with AFLPs and VCGs suggests that sexual reproduction may occur more frequently than estimated by N_e.