Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells.

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.     

TECHNIQUES FOR MEASURING VESSEL LENGTHS AND DIAMETERS IN STEMS OF WOODY PLANTS

Results were compared between the latex paint and compressed air methods for determining total vessel lengths, and between the sectioning and maceration methods for determining vessel diameters. The minimum, mean, median, and maximum vessel diameters were less with the sectioning method than with the maceration technique. Vessel diameter distributions were always nonnormal and had roughly similar patterns with the two techniques, but were statistically different from one another. In all six species where the paint and air methods for determining vessel length were compared, both methods showed a similar skewed vessel length distribution, with many short vessels and few long ones. Although there was no consistent pattern to the difference in results with these two methods, the vessel length frequency distributions were statistically different from one another. With the paint method, many vessels, especially many of the narrowest ones, were not paint-filled at the paint infusion port. The air method utilized the paint method, in part, and, in addition, is based upon the incorrect assumption that all vessels in the stem are the same diameter. Both techniques tended to exclude vessel lengths of the narrowest vessels. However, the narrow vessels, although numerous, contributed an insignificant amount to the total theoretical hydraulic conductance in stems.     

ROOT PRODUCTION IN FOUR COMMUNITIES IN THE GREAT DISMAL SWAMP

A sequential coring approach was used to measure root biomass and production over 1 year in four different communities within the Great Dismal Swamp. A second method, an implanted bag technique, was also used to measure root production, and values were generally lower using this technique. On all sites, fine roots were the most dynamic root component. Both biomass (1,887 g/m²) and production (354–989 g m ² yr^-1) were highest on the mixed hardwood site, the least flooded site, and second highest on the cedar site, the site with the longest duration of soil saturation (1,033 g/m² and 274–366 g m^-2 yr^-1). The maple-gum (696 g/m² and 59–91 g m^-2 yr^-1) and cypress (824 g/m² and 68–308 g m^-2 yr^-1) sites had similarly low amounts of biomass and rates of production. Environmental parameters that influenced production include frequency and duration of flooding, and soil type. Peaks in belowground production were observed on the most productive sites (mixed hardwood and cedar) in summer and late fall-winter; the other two sites exhibited little seasonal variability. The least flooded stand appears to allocate a greater percentage of net primary production belowground than the more extensively flooded stands. The ratio of above- and belowground allocation appears to change in response to a flooding gradient. This has major implications for ecosystem functions as carbon allocation patterns determine the array of litter types generated (leaves vs. roots) which affect decomposition rates and nutrient availability.     

A Rare, Larval-founded Colony of the Bryozoan Archimedes from the Carboniferous of Alabama

Very few basal attachments of the spiralled fenestrate bryozoan Archimedes are known. A newly discovered specimen is interpreted to have grown on a thin cylindrical ephemeral substratum that extended above the sediment-water interface, allowing paired spirals to develop and to extend in opposite directions when the colony was very small.     

Evidence for top predator control of a grazing ecosystem

The importance of top predators in controlling ecological processes in large, intact ecosystems is unclear. In grasslands that support abundant ungulates, top–down control by predators may be particularly important, because of the tight biogeochemical linkages of ungulate prey with plants and soil microbes. Here, I examined the effects of the recent reintroduction of the gray wolf Canis lupus on ecosystem processes in Yellowstone National Park, where herds of grazing ungulates previously have been shown to stimulate several processes, including soil net nitrogen (N) mineralization. Rates of ungulate grazing intensity and soil net N mineralization were compared before and after wolf reintroduction in grasslands ranging five‐fold in aboveground production. Grazing intensity and grassland net N mineralization declined after wolf reintroduction, a likely partial function of fewer ungulates; wolf predation has been one of several factors implicated in causing the decline in Yellowstone ungulates. In addition, the spatial pattern of grazing and net N mineralization changed after reintroduction. A shift in the spatial patterns of grazer‐associated processes is consistent with a growing body of work indicating that wolves have changed habitat use patterns of ungulates in Yellowstone National Park. These findings suggest widespread wolf effects on ungulate prey, plants, and microbial activity that have spatially reorganized grassland energy and nutrient dynamics in Yellowstone Park.     

Biochemical Evidence for Formate Transfer in Syntrophic Propionate-Oxidizing Cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H₂ lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H₂ lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Growth inhibition of putrefactive anaerobe 3679 caused by stringent-type response induced by protonophoric activity of sorbic acid

The inhibitory effects of potassium sorbate on the bioenergetics, phenylalanine uptake, protein synthesis, and certain aspects of cell regulation were examined in putrefactive anaerobe 3679. Undissociated sorbic acid appeared to act as a protonophore by lowering the intracellular pH and dissipating the proton motive force of the membrane. Sorbate inhibited the uptake of phenylalanine, decreased the rate of protein synthesis, and altered patterns of phosphorylated nucleotide accumulation, resulting in increased intracellular concentrations of GTP, ppGpp, and an unidentified compound (possibly pppGpp). The addition of a noninhibitory amount of tetracycline released the inhibition of growth by sorbate. Based on these results, we concluded that the inhibition of putrefactive anaerobe 3679 by sorbate resulted from a stringent-type regulatory response induced by the protonophoric activity of sorbic acid.     

Regulation of the lateral diffusion of WGA-labeled glycoconjugates in human leukocytes. Comparison between adult granulocytes and differentiating promyelocytic HL60 cells

The regulation of the membrane mobility of glycoconjugates in human polymorphonuclear leukocytes (PMNL) was studied by comparing adult PMNL with promyelocytic HL60 cells before and after stimulation of differentiation in HL60 cells with phorbol-myristate acetate (PMA) with respect to lateral diffusion of wheat germ agglutinin (WGA)-labeled glycoconjugates. For this purpose we developed a novel variant of microscope equipment for the study of fluorescence recovery after photobleaching (FRAP) and continuous fluorescence microphotolysis (CFM) using a mini-computer for handling of shutters, data acquisition, and calculations. This equipment is presented in the report. We found that PMA-induced differentiation in HL60 cells reduced the lateral diffusion coefficient (D) of WGA-labeled membrane entities from about 1.5 to 1.0 x 10(-10) cm2/s, which was close to that found for adult blood PMNL, i.e., 1-1.2 x 10(-10) cm2/s. The lateral mobility (D x 10(10)) of succinylated WGA (S-WGA) was 2.3 and 1.7 cm2/s in undifferentiated and PMA-differentiated HL60 cells, respectively, indicating that WGA might have cross-linked membrane receptors, resulting in the slower diffusion. The results are discussed in relation to the effect of phagocyte maturation on the mobility of membrane components.     

Mercury in blood cells—Altered elemental profiles

The study was composed of 27 persons that displayed vague symptoms similar to those of the victims of Minamata and Iraq. Skew distributions of mercury were observed in individual erythrocytes and neutrophil granulocytes when measured by PIXE. Mercury could not be detected in the platelets. The erythrocytes also displayed lowered concentrations of magnesium and zinc, together with increased concentrations of calcium and strontium. The neutrophils displayed decreased concentrations of magnesium and zinc and increased concentrations of calcium, strontium, and iron. The presence of mercury and the altered elemental profiles in the erythrocytes and the neutrophil granulocytes are suggested as early signs of exposure.     

Lipid bilayer polypeptide interactions studied by molecular dynamics simulation

A model membrane with a polypeptide alpha-helix inserted has been simulated by molecular dynamics at a temperature well above the gel/liquid crystalline phase transition temperature. Order parameters of the lipids and other equilibrium and dynamic quantities have been calculated. Three systems, polyglycine constrained into an alphahelical configuration, glycophorin with similarly conformationally constrained backbone and finally glycophorin free to change its backbone conformation, have been studied. In all cases there was an ordering of the chains close to the helix. This effect was, however, much smaller for glycophorin with its rather bulky side chains than for polyglycine. The dynamics of the lipids were affected by the neighbouring helix, not drastically however. Lateral diffusion and reorientational time correlations of lipids close to the helix were slower than for the bulk ones, but not more than two or three times. Thus, we did not find any evidence of bound or frozen boundary lipids.