A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles

Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR-14 and propidium iodide (PI) enabled the discrimination of sperm cells from egg yolk and debris particles, which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm, because it implied that washing steps to remove egg yolk were no longer required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome-damaged/reacted sperm cells from acrosome-intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method-agreement analysis between the SYBR-14/PE-PNA/PI and fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h incubation at 37 degrees C. The British Standards Institution repeatability index of the SYBR-14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple-stain combination is highly repeatable and easy to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.     

A local influence approach applied to binary data from a psychiatric study

Recently, a lot of concern has been raised about assumptions needed in order to fit statistical models to incomplete multivariate and longitudinal data. In response, research efforts are being devoted to the development of tools that assess the sensitivity of such models to often strong but always, at least in part, unverifiable assumptions. Many efforts have been devoted to longitudinal data, primarily in the selection model context, although some researchers have expressed interest in the pattern-mixture setting as well. A promising tool, proposed by Verbeke et al. (2001, Biometrics 57, 43-50), is based on local influence (Cook, 1986, Journal of the Royal Statistical Society, Series B 48, 133-169). These authors considered the Diggle and Kenward (1994, Applied Statistics 43, 49-93) model, which is based on a selection model, integrating a linear mixed model for continuous outcomes with logistic regression for dropout. In this article, we show that a similar idea can be developed for multivariate and longitudinal binary data, subject to nonmonotone missingness. We focus on the model proposed by Baker, Rosenberger, and DerSimonian (1992, Statistics in Medicine 11, 643-657). The original model is first extended to allow for (possibly continuous) covariates, whereafter a local influence strategy is developed to support the model-building process. The model is able to deal with nonmonotone missingness but has some limitations as well, stemming from the conditional nature of the model parameters. Some analytical insight is provided into the behavior of the local influence graphs.     

Effect of yeast extract on speciation and bioavailability of nickel and cobalt in anaerobic bioreactors

The speciation of metals plays an important role in their bioavailability. In the case of anaerobic reactors for the treatment of wastewaters, the ubiquitous presence of sulfide leads to extensive precipitation of metals like nickel and cobalt, which are essential for the metabolism of the anaerobic microorganisms that carry out the mineralization of the pollutants present in the wastewater. In practice, nickel, cobalt, and iron are added in excessive amounts to full-scale installations. This study is concerned with the complexation of nickel and cobalt with yeast extract and its effect on the biogas production by methanogenic biomass. Adsorptive stripping voltammetry (AdSV) was used to get information about the stability and complexing capacity of the metal-yeast extract complexes formed. Nickel and cobalt form relatively strong organic complexes with yeast extract. The bioavailability of these essential metals in anaerobic batch reactors was dramatically increased by the addition of yeast extract. This is due to the formation of dissolved bioavailable complexes, which favors the dissolution of metals from their sulfides. Trace doses of yeast extract may be effective in keeping additions of essential metals to anaerobic reactors at a minimum.     

Carrier screening for Gaucher disease in couples of mixed ethnicity

With the advent of mutational analysis for Gaucher disease, carrier screening has been incorporated into many Jewish genetic disease screening programs. Frequencies and mutations for Gaucher disease in non-Jewish populations are less well established and the detection rate of carriers are lower. Testing is problematic for resolving residual risk in a couple of mixed ethnicity. We report the testing choices made by 20 consecutive couples of mixed ethnicity where the Ashkenazi Jewish partner was identified to be a Gaucher disease gene carrier. Carrier studies of the non-Jewish partner were elected as follows: DNA studies alone, 5 (25%); enzymatic assay, 2 (10%); both, 6 (30%); no carrier studies, 7 (35%). Of the 7 couples not electing carrier studies, one was not in a pregnancy and 6 elected prenatal diagnosis in lieu of parental testing by enzymatic analysis of amniocytes. One couple elected parental carrier studies as well as prenatal diagnosis. All couples electing prenatal Gaucher determination had amniocentesis for other indications as well (4, advanced maternal age; 4, parental anxiety). We conclude that Gaucher screening is feasible for couples of mixed ethnicity if appropriate counseling and testing are offered.     

The dynamics of two diffusively coupled predator-prey populations

I analyze the dynamics of predator and prey populations living in two patches. Within a patch the prey grow logistically and the predators have a Holling type II functional response. The two patches are coupled through predator migration. The system can be interpreted as a simple predator-prey metapopulation or as a spatially explicit predator-prey system. Asynchronous local dynamics are presumed by metapopulation theory. The main question I address is when synchronous and when asynchronous dynamics arise. Contrary to biological intuition, for very small migration rates the oscillations always synchronize. For intermediate migration rates the synchronous oscillations are unstable and I found periodic, quasi-periodic, and intermittently chaotic attractors with asynchronous dynamics. For large predator migration rates, attractors in the form of equilibria or limit cycles exist in which one of the patches contains no prey. The dynamical behavior of the system is described using bifurcation diagrams. The model shows that spatial predator-prey populations can be regulated through the interplay of local dynamics and migration.     

Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi.

"Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patterns and in the parasitic peptides recognition patterns of experimentally infected Wistar rats. The rats experimentally infected with T. evansi isolates obtained from coatis, dogs and horses were submitted to indirect immunofluorescence test (IgM e IgG) and Western blotting. The serological titers for IgM and IgG ranged between 1:40 and 1:160. The most recognized polypeptide profiles were in a range of 17 and 74 kDa. Our data suggest that the humoral immune response in Wistar rats is not sufficient for granting an effective control of T. evansi infections.     

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.     

Mutations in the SAM domain of STE50 differentially influence the MAPK-mediated pathways for mating, filamentous growth and osmotolerance in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the MAPKKK Ste11p is involved in three mitogen-activated protein kinase (MAPK) pathways required for mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. All three pathways are also dependent on Ste50p. Ste50p and Ste11p interact constitutively via their N-terminal regions, which include putative SAM domains. Here we show that the interaction of Ste50p and Ste11p is differentially required for modulation of Ste11p function during mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. Two derivatives of Ste50p with mutations in the SAM domain were isolated and characterised. The mutant Ste50 proteins showed reduced binding to Ste11p and a tendency to form homodimers in two-hybrid and in vitro binding assays. Interestingly, these two Ste50p-SAM mutants were associated with increased activation of the mating and filamentous-growth pathways, but a reduction in the SHO1-dependent growth response to hyperosmolarity, relative to the wild-type Ste50p. Moreover, when exposed to hyperosmolarity, these Ste50p-SAM mutants activate genes in the mating (FUS1) and filamentous-growth (FLO11) pathways to higher levels than does the wild type. Thus the Ste50p-Ste11p interaction may differentially modulate the flow of information through the various MAPK-mediated pathways.     

The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.