Immobilization Antigens from Tetrahymena thermophila Are Glycosyl-Phosphatidylinositol-Linked Proteins

We have studied four strains of Tetrahymena thermophila , each of which expresses a different allele of the SerH gene and produces a distinctive surface protein of the immobilization antigen (i-antigen) class. Following exposure of the strains to [³H]ethanolamine or [³H]myristic acid, a protein corresponding in molecular mass to the characteristic i-antigen for that strain became highly labeled, as determined by mobility in sodium dodecylsulfate-polyacrylamide eiectrophoresis gels. Furthermore, antibodies raised to the i-antigens of the T. thermophila strains selectively immunoprecipitated radioactive proteins having molecular mass identical to that of the i-antigen characteristic for that particular strain. The lipid moieties labeled by [³H]myristate were not susceptible to hydrolysis by exogenous phosphatidylinositol-specific phospholipase C from bacteria. However, when protein extraction was carried out in the absence of phospholipase C inhibitors, radioactive fatty acids derived from [³H]myristate were rapidly cleaved from the putative i-antigens. On the basis of available data, it was concluded that T. thermophila i-antigens contain covalently-Iinked glycosyl-phospha-tidylinositol anchors.     

Titin: quantitative mass measurements by scanning transmission electron microscopy and structural implications for the sarcomere matrix of skeletal muscle

Scanning transmission electron microscopy has been used to investigate mass and linear mass density of native titin-2, a large soluble fragment of intact titin, from rabbit skeletal muscle. Dark field images of unstained, freeze-dried titin-2 appeared as either compact globules or looser and larger balls of string. Direct mass measurements indicated that the compact forms have an average mass of 2.40 +/- 0.50 x 10(6) Da. The mass to length ratio, determined from well-spread portions of titin strands (3-5 nm wide) from the ball of string forms, averaged 2.7 +/- 0.9 kDa/nm. Thus a single native intact titin molecule has a calculated contour length of well above approximately 1 micron, sufficient to span unidirectionally between the Z line and M line region in a resting-length sarcomere.     

Role of Motility and Chemotaxis in Efficiency of Nodulation by Rhizobium meliloti

Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants was able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation in the initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10⁵ to 10⁶ bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants. The mutants were also somewhat less effective than their parent as competitors in root adsorption assays. It appears that motility and chemotaxis are quantitatively important traits that facilitate the initial contact and adsorption of symbiotic rhizobia to the host root surface, increase the efficiency of nodule initiation, and increase the rate of infection development.     

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamical Model of Drug Accumulation in Bacteria: Sensitivity Analysis and Experimentally Testable Predictions

We present a dynamical model of drug accumulation in bacteria. The model captures key features in experimental time courses on ofloxacin accumulation: initial uptake; two-phase response; and long-term acclimation. In combination with experimental data, the model provides estimates of import and export rates in each phase, the time of entry into the second phase, and the decrease of internal drug during acclimation. Global sensitivity analysis, local sensitivity analysis, and Bayesian sensitivity analysis of the model provide information about the robustness of these estimates, and about the relative importance of different parameters in determining the features of the accumulation time courses in three different bacterial species: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The results lead to experimentally testable predictions of the effects of membrane permeability, drug efflux and trapping (e.g., by DNA binding) on drug accumulation. A key prediction is that a sudden increase in ofloxacin accumulation in both E. coli and S. aureus is accompanied by a decrease in membrane permeability.     

Negative fire feedback in a transitional forest of southeastern Amazonia

Anthropogenic understory fires affect large areas of tropical forest, particularly during severe droughts. Yet, the mechanisms that control tropical forests' susceptibility to fire remain ambiguous. We tested the widely accepted hypothesis that Amazon forest fires increase susceptibility to further burning by conducting a 150 ha fire experiment in a closed-canopy forest near the southeastern Amazon forest–savanna boundary. Forest flammability and its possible determinants were measured in adjacent 50 ha forest plots that were burned annually for 3 consecutive years (B3), once (B1), and not at all (B0). Contrary to expectation, an annual burning regime led to a decline in forest flammability during the third burn. Microclimate conditions were more favorable compared with the first burn (i.e. vapor pressure deficit increased and litter moisture decreased), yet flame heights declined and burned area halved. A slight decline in fine fuels after the second burn appears to have limited fire spread and intensity. Supporting this conclusion, fire spread rates doubled and burned area increased fivefold in B3 subplots that received fine fuel additions. Slow replacement of surface fine fuels in this forest may be explained by (i) low leaf litter production (4.3 Mg ha⁻¹ yr⁻¹), half that of other Amazon forests; and (ii) low fire-induced tree and liana mortality (5.5±0.5% yr⁻¹, SE, in B3), the lowest measured in closed-canopy Amazonian forests. In this transitional forest, where severe seasonal drought removed moisture constraints on fire propagation, a lack of fine fuels inhibited the intensity and spread of recurrent fire in a negative feedback. This reduction in flammability, however, may be short-lived if delayed tree mortality or treefall increases surface fuels in future years. This study highlights that understanding fuel input rate and timing relative to fire frequency is fundamental to predicting transitional forest flammability – which has important implications for carbon emissions and potential replacement by scrub vegetation.