Sperm velocity and its relation to social status in Arctic charr (Salvelinus alpinus)

Sperm velocity has been shown to be a major determinant of fertilization success of external fertilizers. Yet, sperm velocity varies both within and between ejaculates and only a small number of fast sperm cells within an ejaculate are likely to have the potential of fertilizing the eggs. Having such fast cells should be of special importance during sperm competition, particularly for subordinate males that may release their sperm later or further away from eggs, than dominants. We examined ejaculates of dominant and subordinate male Arctic charr (Salvelinus alpinus), a species with sperm competition. Yet, rather than examining just average sperm velocity values, the aim was to examine whether the fastest fraction of sperm cell from dominant and subordinate males differed in velocity. While there was no difference in the average sperm velocity between dominant and subordinate males, analysis of the fastest swimming sperm cells show that subordinate males have significantly higher initial sperm velocity than dominant males within the 10, 5 and 1% fastest sperm cells. That is, the difference in sperm velocity between dominant and subordinate charr is most predominant among the fastest sperm cells. In sum, this study emphasizes the importance of studying the fastest sperm cells in the ejaculates, as status-dependent differences in sperm velocity may not be detected using average values.     

Occasional intraguild predation structuring small mammal assemblages: the marsupial Didelphis aurita in the Atlantic Forest of Brazil

The didelphid marsupial, Didelphis aurita, is suggested as an intraguild predator and as key‐species in small mammal assemblages of the Atlantic Forest of Brazil. The field experiments required to test this hypothesis are complex to implement, but the recent revival of regression methods offers a viable alternative. Here we use the dynamic and static regression methods to determine the importance of D. aurita as a competitor and intraguild predator. Capture–recapture data from two localities in the Rio de Janeiro State were used, Garrafão (municipality of Guapimirim), a coastal forest of the Serra do Mar, and Barra de Maricá, a costal sand dune vegetation. Population and microhabitat variables were monitored from April 1997 to April 2003 in Garrafão, and from January 1986 to July 1990 in Barra de Maricá. Microhabitat variables were related to Canopy, Plant, Litter and Rock covers, Obstruction from 0 to 1.5 m, and Number of logs. Exploitation competition was tested by the dynamic method, which models the effects of D. aurita on the per capita growth rate of a species. Interference by predation or competition was tested by the static method, where the abundance of D. aurita at trap stations was regressed against the abundance of other small mammals, after removal of any variation associated with microhabitat factors. Exploitation competition was not detected, but the interference of D. aurita was pervasive, affecting all small mammals studied in the two localities. The clear avoidance of D. aurita by all small mammals tested in two localities of different physiognomies indicates that it functions as an intraguild predator, even if actual predation by D. aurita is an occasional event.     

toxin-mediated insect resistance in plants

We are currently in an interesting phase of plant biotechnology releases, both for the scientists responsible for these innovations who are beginning to see their ideas realized, and for the biotechnology companies that are starting to see a return on their investment. One of the most notable examples, is the introduction of transgenic crops that are engineered to express a Bacillus thuringiensis toxin that confers resistance to insect predation. However, the picture is not altogether positive - there is concern that the introduction of this technology was premature or should not have happened at all, and that the valuable insecticidal properties of Bacillus thuringiensis will be lost.     

Evaluation of a rapid,quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.     

Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution

We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.     

Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species

A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of selected Aspergillus, Penicillium and Paecilomyces species. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.     

Oscillatory pericellular proteolysis and oxidant deposition during neutrophil locomotion.

To better understand the mechanism of leukocyte migration in complex environments, model extracellular matrices were prepared using gelatin, Hanks' solution, Bodipy-BSA (fluorescent upon proteolysis), and dihydrotetramethylrosamine or hydroethidine (fluorescent upon oxidation). Using quantitative microfluorometry, neutrophil-mediated extracellular pulses of reactive oxygen metabolites (ROMs) and pericellular proteolysis were periodically observed showing that these functions occur as quantal bursts. However, chronic granulomatous disease neutrophils, which do not produce ROMs, did not display ROM deposition. Matrices show an alternating pattern of green (proteolytic) and red (oxidative) fluorescence, indicating these functions are out of phase. Electric fields phase-matched with metabolic oscillations, which increase the amplitude of intracellular NAD(P)H oscillations, increase ROM deposition and pericellular proteolysis; this further supports the link between intracellular chemical oscillators and extracellular functions. This phase relationship may allow ROMs to inactivate protease inhibitors, followed by protease activation.     

Structural characterization and independent folding of a chimeric glycoprotein comprising granulocyte-macrophage colony stimulating factor and erythropoietin sequences

MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.