Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (<Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Ait.)

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar ‘HyRed’. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540–590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2–15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.     

A shotgun proteomic method for the identification of membrane-embedded proteins and peptides

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome.     

Induction of differentiation of murine embryonal carcinoma cells by ouabain

Murine embryonal carcinoma cells (EC) can be induced to differentiate by a variety of chemical agents, including retinoid acid (RA) and dimethyl acetamide (DMA). However, it is not known how these agents exert their effects. In this study we demonstrate that murine EC cells can also be induced to differentiate by ouabain at concentrations which inhibit Na+, K+-ATPase activity as measured by inhibition of 86Rb+ uptake. Since the pharmacologic action of ouabain is thought to be specific, we investigated the role of Na+, K+-ATPase inhibition and specific metabolic consequences of this inhibition in the induction of EC differentiation, and explored whether this might be a common mode of action for a variety of structurally diverse inducers. Although the Na+, K+-ATPase maintains ion gradients in cells, our studies failed to demonstrate a consistent role for alterations of ion flux or ion concentration on the differentiation process. Ouabain inhibited cell growth, but a direct correlation between the degree of growth inhibition and the extent of differentiation could not be demonstrated. There was also no evidence that RA or DMA induces differentiation by inhibiting the Na+, K+-ATPase. The mechanism of ouabain induction may be mediated by some alternative consequence of Na+, K+-ATPase inhibition, but it appears to be specific for that inducer and cannot be generalized to that of other inducers of EC differentiation.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF ?ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF ?ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.     

Nucleolar persistence in embryonal carcinoma cells

A series of embryonal carcinoma (EC) lines were found to have nucleoli which persisted through metaphase and anaphase in 60–88% of cells. When these cells differentiated, either spontaneously or upon chemical induction with retinoic acid or dimethylacetamide, the level of nucleolar persistence dropped to under 20%. Mouse embryo fibroblasts and mouse bone marrow cells had few persistent nucleoli. Persistent nucleoli in EC cells contained DNA, labeled with tritiated uridine and stained with ammoniacal silver, all of which support the hypothesis that rRNA synthesis continues in persistent nucleoli.     

Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.     

Anxiety‐related behavioral inhibition in rats: a model to examine mechanisms underlying the risk to develop stress‐related psychopathology

Behavioral inhibition (BI) is an adaptive defensive response to threat; however, children who display extreme BI as a stable trait are at risk for development of anxiety disorders and depression. The present study validates a rodent model of BI based on an ethologically relevant predator exposure paradigm. We show that individual differences in rat BI are stable and trait‐like from adolescence into adulthood. Using in situ hybridization to quantify expression of the immediate early genes homer1a and fos as measures of neuronal activation, we show that individual differences in BI are correlated with the activation of various stress‐responsive brain regions that include the paraventricular nucleus of the hypothalamus and CA3 region of the hippocampus. Further supporting the concept that threat‐induced BI in rodents reflects levels of anxiety, we also show that BI is decreased by administration of the anxiolytic, diazepam. Finally, we developed criteria for identifying extreme BI animals that are stable in their expression of high levels of BI and also show that high BI (HBI) individuals exhibit maladaptive appetitive responses following stress exposure. These findings support the use of predator threat as a stimulus and HBI rats as a model to study mechanisms underlying extreme and stable BI in humans.