Immunochemical studies of Aspergillus fumigatus mycelial antigens by polyacrylamide gel electrophoresis and western blotting techniques

Differences were detectable among strains of the opportunist fungal pathogen Aspergillus fumigatus when water-soluble (WS) preparations were analysed by combined SDS-PAGE and Western blotting procedures. A wide range of molecules of apparent molecular masses from approximately 20 to greater than 100 kDa showed specific binding to antibodies raised in rabbits to A. fumigatus wall and cytoplasmic components. The ability to bind antibody was markedly reduced by treatment of these antigens with sodium periodate or with specific proteases or glucanases. Pretreatment of blotted antigens with either concanavalin A (ConA) or wheat germ agglutinin (WGA) did not, however, inhibit subsequent antibody binding. The antigens of subfractions prepared from a single strain of A. fumigatus WS material were also susceptible to periodate oxidation and enzymic hydrolysis. Slight cross-reactivity was apparent when crude preparations of cellular or culture filtrate antigens, used in this laboratory to detect antibodies to Candida albicans, Coccidioides immitis and Cryptococcus neoformans, were probed with hyperimmune rabbit antisera to A. fumigatus. Efforts were made to characterize the WS preparations of A. fumigatus, used as diagnostic antigens in many laboratories. The electrophoretically separated antigenic moieties were shown to be predominantly glycoproteins. Binding of cytoplasmic antigens to antibodies raised to wall material showed the presence of many common components in both wall and cytosol. Antiserum to wall components revealed most differentiation among A. fumigatus strains.     

'Hot spot' amino acid distribution in Ha-ras oncogene product p21: relationship to guanine binding site.

Although the rules which describe the atomic basis of structure-function relationships of proteins have yet to be deciphered, they are nevertheless coded within the framework of the amino acid and nucleotide sequence. The objectives of the present investigations were to document a composite, new approach for the evaluation of the structure-function dependencies of proteins based on the analysis of the informational content of the primary amino acid sequence as well as the topological and functional regions of a protein. This approach is validated with the example of the p21 Ha-ras oncogene family of proteins. Using this approach, amino acids crucial for p21 transforming activity have been identified and these amino acid residue assignments compared with experimental data.     

Real-time quantitation of viral replication and inhibitor potency using a label-free optical biosensor

Real-time detection of viral replication inside cells remains a challenge to researchers. The Epic^® System is a high-throughput, label-free optical detection platform capable of measuring molecular interaction in a biochemical assay, as well as integrated cellular response from measurement of cellular dynamic mass redistribution (DMR) in a cell-based assay. DMR has previously been used to measure cell signaling upon receptor stimulation. In this report, we present the first example of Epic^® measurement of viral replication-induced cellular response and demonstrate that this system is extremely powerful not only for the sensitive and quantitative detection of viral replication inside cells but also for screening of viral inhibitors. By comparing with conventional assays used for the measurement of viral replication, we show that the Epic^® response has many advantages including sensitivity, high throughput, real-time quantification and label-free detection. We propose that the Epic^® system for measurement of integrated cellular response will be an excellent method for elucidating steps in viral replication as well as for the high-throughput screening of inhibitors of rhinovirus and other viruses.     

Diel movements of out-migrating Chinook salmon (Oncorhynchus tshawytscha) and steelhead trout (Oncorhynchus mykiss) smolts in the Sacramento/San Joaquin watershed

We used ultrasonic telemetry to describe the movement patterns of late-fall run Chinook salmon (Oncorhynchus tshawytscha) and steelhead trout (O. mykiss) smolts during their entire emigration down California’s Sacramento River, through the San Francisco Bay Estuary and into the Pacific Ocean. Yearling hatchery smolts were tagged via intracoelomic surgical implantation with coded ultrasonic tags. They were then released at four upriver locations in the Sacramento River during the winters of 2007 through 2010. Late-fall run Chinook salmon smolts exhibited a nocturnal pattern of migration after release in the upper river. This is likely because individuals remain within a confined area during the day, while they become active at night and migrate downstream. The ratio between night and day detections of Chinook salmon smolts decreased with distance traveled downriver. There was a significant preference for nocturnal migration in every reach of the river except the Estuary. In contrast, steelhead smolts, which reside upriver longer following release, exhibited a less pronounced diel pattern during their entire migration. In the middle river, Delta, and Estuary, steelhead exhibited a significant preference for daytime travel. In the ocean Chinook salmon preferred to travel at night, yet steelhead were detected on the monitors equally during the night and day. These data show that closely related Oncorhynchus species, with the same ontogenetic pattern of out-migrating as yearlings, vary in migration tactic.     

Low‐coverage genomic data resolve the population divergence and gene flow history of an Australian rain forest fig wasp

Population divergence and gene flow are key processes in evolution and ecology. Model‐based analysis of genome‐wide data sets allows discrimination between alternative scenarios for these processes even in nonmodel taxa. We used two complementary approaches (one based on the blockwise site frequency spectrum [bSFS], the second on the pairwise sequentially Markovian coalescent [PSMC]) to infer the divergence history of a fig wasp, Pleistodontes nigriventris. Pleistodontes nigriventris and its fig tree mutualist Ficus watkinsiana are restricted to rain forest patches along the eastern coast of Australia and are separated into The Northern population is to the north of the Southern populations by two dry forest corridors (the Burdekin and St. Lawrence Gaps). We generated whole genome sequence data for two haploid males per population and used the bSFS approach to infer the timing of divergence between northern and southern populations of P. nigriventris, and to discriminate between alternative isolation with migration (IM) and instantaneous admixture (ADM) models of postdivergence gene flow. Pleistodontes nigriventris has low genetic diversity (π = 0.0008), to our knowledge one of the lowest estimates reported for a sexually reproducing arthropod. We find strongest support for an ADM model in which the two populations diverged ca. 196 kya in the late Pleistocene, with almost 25% of northern lineages introduced from the south during an admixture event ca. 57 kya. This divergence history is highly concordant with individual population demographies inferred from each pair of haploid males using PSMC. Our analysis illustrates the inferences possible with genome‐level data for small population samples of tiny, nonmodel organisms and adds to a growing body of knowledge on the population structure of Australian rain forest taxa.     

Extreme environments select for reproductive assurance: evidence from evening primroses (Oenothera)

Competing evolutionary forces shape plant breeding systems (e.g. inbreeding depression, reproductive assurance). Which of these forces prevails in a given population or species is predicted to depend upon such factors as life history, ecological conditions, and geographical context. Here, we examined two such predictions: that self-compatibility should be associated with the annual life history or extreme climatic conditions. We analyzed data from a clade of plants remarkable for variation in breeding system, life history and climatic conditions (Oenothera, sections Anogra and Kleinia, Onagraceae). We used a phylogenetic comparative approach and Bayesian or hybrid Bayesian tests to account for phylogenetic uncertainty. Geographic information system (GIS)-based climate data and ecological niche modeling allowed us to quantify climatic conditions. Breeding system and reproductive life span are not correlated in Anogra and Kleinia. Instead, self-compatibility is associated with the extremes of temperature in the coldest part of the year and precipitation in the driest part of the year. In the 60 yr since this pattern was anticipated, this is the first demonstration of a relationship between the evolution of self-compatibility and climatic extremes. We discuss possible explanations for this pattern and possible implications with respect to anthropogenic climate change.     

Identification of Tf1 integration events in S. pombe under nonselective conditions

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning the integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the Schizosaccharomyces pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418^S/neo⁺ Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418^S/neo⁺ clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1's ability to insert within silent regions of S. pombe's genome.     

N‐Heterocyclic‐based adsorbents for antibody purification—effect of ligand structure

A new set of ligands based on substituted pyridine and other N‐heterocyclic structures, possessing an aliphatic primary amino group tether and an exocyclic sulphur atom, has been prepared and immobilized onto epoxy‐activated matrices such as Sepharose 6 Fast Flow®. The derived adsorbents have been evaluated for their utility to capture and purify humanized monoclonal antibodies. Favourable binding properties were assessed from screening assays to determine optimal conditions for the capture and elution of the monoclonal antibodies. Static and dynamic binding experiments were employed to derive the equilibrium dissociation constants K_D's and binding capacities Q_max's. Typically, the K_D values were in the range of 2–5 μM and the Q_max values between 20 and 75 mg mAb/ml resin, depending on the stereo‐electronic properties of the substituent in the N‐heterocyclic ring structure. The effect of ligand structure on the selectivity of these adsorbents was also investigated, and criteria for their use in the purification of monoclonal antibodies from cell culture supernatants established. Copyright © 2014 John Wiley & Sons, Ltd.