Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

Pacific Coral-reef Fishes: The Implications of Behaviour and Ecology of Larvae for Biodiversity and Conservation, and a Reassessment of the Open Population Paradigm

The two-phase life history of most marine fishes and invertebrates has enormous implications for dispersal, population connectivity, and resource management. Pelagic dispersal larvae of marine animals traditionally thought to ensure that populations are widespread, that chances of local extinction are low, and that marine protected areas (MPA) can easily function to replenish both their own populations and those of unprotected areas. Traditionally, dispersal is considered to depend primarily on two variables: pelagic larva duration and far-field currents. These conclusions arise from the open population paradigm and are usually accompanied by a simplifying assumption: larvae are distributed passively by far-field currents. Unfortunately, they ignore the complex reality of circulation and hydrological connectivity of reefs, and do not consider newly-demonstrated behavioural capabilities of coral-reef fish larvae. Far-field circulation varies with depth and often excludes water bodies where propagules are released, and this has important implications for predicting trajectories of even passive larvae. However, larvae are not passive: late-stage larvae of coral-reef fishes can swim faster than currents for long periods, can probably detect reefs at some distance, and can actively find them. This behaviour is flexible, which greatly complicates modelling of larval fish trajectories. Populations at ecological (as opposed to evolutionary) scales are probably less open and more subdivided than previously assumed. All this means that dispersal predictions based solely on far-field water circulation are probably wrong. An emerging view of larval-fish dispersal is articulated that takes these new data and perspectives into account. This emerging view shows that re-evaluation of traditional views in several areas is required, including the contribution of larval-fish biology and dispersal to biodiversity patterns, the way reef fishes are managed, and the way in which MPA are thought to operate. At evolutionary and zoogeographic scales, reef-fish populations are best considered to be open.     

Secretion of fucosylated oligosaccharides related to the H antigen by human gastric cells

 Although the role of the blood group antigens in the gastrointestinal tract is not well understood, alterations in blood group-related antigens have been described in some pathological processes. Thus, the knowledge of their expression under normal conditions is of special interest. Those individuals expressing their ABO blood group in exocrine epithelia and secretions are called secretors. The aim of the present study was the localization of H antigen expression in the normal human gastric epithelial cells of non-O blood group individuals. For this, a monoclonal anti-H antibody was examined by immunocytochemical methods at both the light and electron microscopic levels. In combination with enzymatic and chemical treatments, the nature of the oligosaccharide chains containing the H antigen was characterized. The selected cases were four A secretors, three A non-secretors, and three B non-secretors. The labeling of the anti-H antibody in the human stomach is described, irrespective of the blood group of the individuals. The staining was abolished when O-linked oligosaccharides were removed. Since commercially available anti-H antibodies usually also recognize other H-related antigens, the labeling of the antibody by H-related antigens cannot be dismissed. Our findings suggest the existence of H or H-related antigens in the O-linked oligosaccharides of the secretory granules of the surface, gastric pit, mucous neck, and transitional cells of the fundic mucosa, and in the intracellular canaliculi and tubulovesicular system of parietal cells. The H or H-related antigens were also localized in the apical membrane of all the cell types of the epithelial cells of the human fundic mucosa. The overall distribution of the H or H-related antigens in the stomach in non-O blood group individuals suggests the constitutive expression of an α(1,2)fucosyltransferase. Received: 24 October 1997 / Accepted: 3 March 1998     

Gas chromatography-negative ion chemical ionisation mass spectrometry using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl derivatives for the quantitative determination of methylphenidate in human plasma

A novel electrophoric derivatisation procedure using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride for the quantitative determination of methylphenidate in human plasma is described. The drug can be quantitatively measured down to 0.006 pg/mL plasma due to the extraordinary sensitivity of the derivatives under negative ion chemical ionisation mass spectrometry. Plasma samples were made alkaline with carbonate buffer and treated with extraction solvent (n-hexane) and reagent solution for 15 min, which, after concentration was measured by GC-NICI-MS. The method is rapid as extraction and derivatisation occur in one single step. A stable isotope labelled internal standard was used. Validation data are given to demonstrate the usefulness of the assay, including selectivity, linearity, accuracy and precision, autosampler stability, aliquot analysis, robustness, and prospective analytical batch size accuracy. The method has been successfully applied to pharmacokinetic profiling of the drug after oral administration.     

A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi

Background  

Gene loss, inversions, translocations, and other chromosomal rearrangements vary among species, resulting in different rates of structural genome evolution. Major chromosomal rearrangements are rare in most eukaryotes, giving large regions with the same genes in the same order and orientation across species. These regions of macrosynteny have been very useful for locating homologous genes in different species and to guide the assembly of genome sequences. Previous analyses in the fungi have indicated that macrosynteny is rare; instead, comparisons across species show no synteny or only microsyntenic regions encompassing usually five or fewer genes. To test the hypothesis that chromosomal evolution is different in the fungi compared to other eukaryotes, synteny was compared between species of the major fungal taxa.     

In vitro engagement of CD3 and CD28 corrects T cell defects in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells concomitant with immunological abnormalities and depressed immune responses. The T cell abnormalities found in CLL patients are thought to increase the risk of infection and hamper immune recognition and elimination of leukemic cells. We evaluated whether providing signals through CD3 and CD28 would correct some of these T cell defects. PBMC were incubated with anti-CD3 and anti-CD28 mAbs conjugated to superparamagnetic beads for 12-14 days. This resulted in a 1400-fold increase in T cell numbers. Activated T cells expressed high levels of CD25, CD54, CD137, and CD154, and produced IFN-gamma, TNF-alpha, and GM-CSF. The mean T cell composition of cultures increased from approximately 6% to >90% and leukemic B cells decreased from a mean of approximately 85% to 0.1% or less. Leukemic B cells up-regulated expression of CD54, CD80, CD86, and CD95. Receptor up-regulation required direct cell contact with the activated T cells and could be blocked with anti-CD154 mAb, suggesting that the CD40-CD40L pathway helped mediate these effects. Poor T cell responses to allostimulation were corrected by the activation and expansion process. The skewing in the TCR repertoire returned to normal, or near normal following the culture process in eight of nine patients with abnormal TCR repertoires. Activated T cells had potent in vitro antileukemic effects in contrast to nonactivated T cells. Based upon these findings, a clinical trial has been initiated to test the potential therapeutic effects of T cells activated using this approach in patients with CLL.     

Using the realized relationship matrix to disentangle confounding factors for the estimation of genetic variance components of complex traits

Background

In the analysis of complex traits, genetic effects can be confounded with non-genetic effects, especially when using full-sib families. Dominance and epistatic effects are typically confounded with additive genetic and non-genetic effects. This confounding may cause the estimated genetic variance components to be inaccurate and biased.

Methods

In this study, we constructed genetic covariance structures from whole-genome marker data, and thus used realized relationship matrices to estimate variance components in a heterogenous population of ~ 2200 mice for which four complex traits were investigated. These mice were genotyped for more than 10,000 single nucleotide polymorphisms (SNP) and the variances due to family, cage and genetic effects were estimated by models based on pedigree information only, aggregate SNP information, and model selection for specific SNP effects.

Results and conclusions

We show that the use of genome-wide SNP information can disentangle confounding factors to estimate genetic variances by separating genetic and non-genetic effects. The estimated variance components using realized relationship were more accurate and less biased, compared to those based on pedigree information only. Models that allow the selection of individual SNP in addition to fitting a relationship matrix are more efficient for traits with a significant dominance variance.     

Drosophilid Assemblages at Different Urbanization Levels in the City of Porto Alegre, State of Rio Grande do Sul, Southern Brazil

The present study analyzed the drosophilid assemblages in different levels of urbanization in the city of Porto Alegre, Rio Grande do Sul, Brazil. Collections were carried out in 2008 in three different environments: a highly urbanized area????Jardim Botanico,?? a forested area with intermediary urbanization????Parque Gabriel Knijnik,?? and in a relatively well-preserved forested area, although threatened by the urban growth????Morro Santana.?? In Jardim Botanico, 36 species belonging to four genera were found, with high abundance of exotic species as Drosophila simulans Sturtevant and Zaprionus indianus (Gupta). In Parque Gabriel Knijnik, 33 species that belonged to four genera were found, with higher abundances of native species belonging to the Drosophila tripunctata species group and Drosophila willistoni species subgroup, and lower abundance of exotic species. As for Morro Santana, 32 species and three genera were found, with higher abundances of native groups, low representativeness of exotic species, and absence of Zaprionus indianus. The analysis of the Jaccard index showed higher similarity in the species composition between samples collected in summer and autumn, and between samples collected in winter and spring. On the other hand, the Morisita index differentiated Jardim Botanico from the other two studied sites. Our results show that Morro Santana is an important area of native biodiversity, reinforcing, therefore, the inclusion of this area in the project for the creation of an ecological corridor as proposed by the Ministry of the Environment of Brazil.