Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ± 3.7% [mean ± standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r² = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.     

Eimeria acervulina: cloning of a cDNA encoding an immunogenic region of several related merozoite surface and rhoptry proteins.

A cDNA encoding a recombinant Eimeria acervulina antigen, designated EAMZp30-47, that contains an epitope shared among several surface and rhoptry proteins of merozoites was characterized. The respective parasite proteins are between 30 and 47 kDa as revealed by immunostaining of nitrocellulose membrane containing extracts of 125I-labeled merozoites. As indicated by immunofluorescence and immunoelectron microscopic staining, the reactive epitope was localized to both the surface membrane and the internal rhoptries of this asexual stage of the parasite. The recombinant beta-galactosidase fusion protein EAMZp30-47 is 130 kDa, thus representing 15 kDa or 30-50% of the respective parasite protein. Purified EAMZp30-47 stimulates T cells from E. acervulina-immune inbred chickens, but is not recognized by immune chicken serum, suggesting that T cell and not B cell epitopes recognized by the host immune system during a natural infection are present on the recombinant protein. Northern and Southern blot hybridization experiments indicated that expression of EAMZp30-47 is restricted to the merozoite stage of the parasite and the gene occurs as a single copy sequence within the genome.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Are all GMOs the same? Consumer acceptance of cisgenic rice in India

India has more than 215 million food‐insecure people, many of whom are farmers. Genetically modified (GM) crops have the potential to alleviate this problem by increasing food supplies and strengthening farmer livelihoods. For this to occur, two factors are critical: (i) a change in the regulatory status of GM crops, and (ii) consumer acceptance of GM foods. There are generally two classifications of GM crops based on how they are bred: cisgenically bred, containing only DNA sequences from sexually compatible organisms; and transgenically bred, including DNA sequences from sexually incompatible organisms. Consumers may view cisgenic foods as more natural than those produced via transgenesis, thus influencing consumer acceptance. This premise was the catalyst for our study—would Indian consumers accept cisgenically bred rice and if so, how would they value cisgenics compared to conventionally bred rice, GM‐labelled rice and ‘no fungicide’ rice? In this willingness‐to‐pay study, respondents did not view cisgenic and GM rice differently. However, participants were willing‐to‐pay a premium for any aforementioned rice with a ‘no fungicide’ attribute, which cisgenics and GM could provide. Although not significantly different (P = 0.16), 76% and 73% of respondents stated a willingness‐to‐consume GM and cisgenic foods, respectively.     

Gonadotropin-releasing hormone infusion interval: importance in pharmacodynamics

Circulating gonadotropin-releasing hormone (GnRH) levels were measured during and after intravenous infusion intervals ranging from 0.08 minutes to 5 minutes and doses ranging from 1 to 25 micrograms per pulse. In all dose groups (1 vs. 5 vs. 25 micrograms), the peak levels of GnRH decreased from the 0.08 minute to the 5 minute infusion interval. Our results suggest that the infusion interval over which GnRH is administered has profound effects on the amount, duration, and pattern of GnRH measured in the peripheral circulation.     

Free-flow electrophoretic separation of Plasmodium berghei sporozoites

Sporozoites of the rodent malaria, Plasmodium berghei, were obtained from infected Anopheles stephensi by grinding mosquitoes, prepurifying the material in a discontinuous Hypaque gradient and further purifying by means of continuous free-flow electrophoresis. Bacteria, debris, mitochondria, mitoplasts, and other contaminants were removed in the electric field. The isolated sporozoites were morphologically intact and were positive in indirect immunofluorescence assay. They were infective to mice prior to and following free-flow electrophoretic separation. The surface of the sporozoites exhibited a polysaccharide-rich layer. The predominant surface protein labelled after surface iodination had a molecular weight between 42,000 and 46,000 daltons.     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.