Lectin histochemistry of nephroblastoma (Wilms' tumour)

Summary Paraffin sections of seven cases of nephroblastoma and one case of clear cell sarcoma were stained with a battery of eleven lectins conjugated to horseradish peroxidase. Lectin staining revealed similarities between blastema and stroma with respect to their content of glycoconjugates whereas blastema and epithelial cells exhibited major differences. In general, blastema and stroma contained glycoconjugates with terminal or penultimate -galactose, glycoconjugates having either biantennary or triantennary N-linked sugar chains or both, sialoglycoconjugates, and occasionally glycogen. Epithelial cells also showed these complex carbohydrates but stained additionally for terminal disaccharide galactose-(13)-N-acetylgalactosamine, terminal -galactose and terminal -N-acetylgalactosamine. Furthermore, staining with three fucose-binding lectins revealed that the linkage between terminal -fucose residues to the constituent oligosaccharide chains varied between epithelial cells, blastema and stroma. In general, the distribution and content of glycoconjugates in tumour cells comprising clear cell sarcoma resembled that in blastema and stroma of nephroblastoma. Other findings included differences in content of glycosubstance between cuboidal and columnar cells within the same tumour. Also observed were variations between a primary tumour and its metastasis with respect to the occurrence of certain complex carbohydrates.     

Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.     

Temporal and topological clustering of diverged residues among enterobacterial dihydrofolate reductases.

The complete nucleotide and encoded amino acid sequences were determined for the dihydrofolate reductase (DHFR) from the bacteria Enterobacter aerogenes and Citrobacter freundii. These were compared with the closely related Escherichia coli DHFR sequence. The ancestral DHFR sequence common to these three species was reconstructed. Since that ancestor there have been seven, nine, and one amino acid replacements in E. coli, E. aerogenes, and C. freundii, respectively. In E. coli, five of its seven replacements were located in the beta-sheet portion of the protein, and all seven were located in a single restricted region of the protein. In E. aerogenes, all nine of its replacements were located within surface residues, with five clustered in a region topologically distinct from the E. coli cluster. The replaced side chains are sometimes in direct contact but more often are separated by an intervening side chain. It is argued that the temporal clustering of replacements is typical for the evolution of most proteins and that the associated topological clustering gives a picture of how evolutionary change is accommodated by protein structure.     

Avian hematozoa from west-central Bolivia

A total of 641 birds representing 135 species of 25 families from Noel Kempff Mercado National Park in west-central Bolivia was examined for hematozoa; only 33 (5.1%) harbored blood parasites. Microfilariae were the most commonly encountered hematozoans, followed, in numerical sequence, by species of Haemoproteus and Plasmodium; Trypanosoma, Atoxoplasma, and Hepatozoon were seen infrequently. The survey included 13 new host-parasite records, and 58 species of birds were examined for blood parasites for the first time; 43 were parasite-free. The low prevalence of parasitism recorded in this survey is compared to other areas in the Neotropical region and to prevalence of blood parasites in the avifauna of other major land masses.     

Identification of Grandchildless Loci Whose Products Are Required for Normal Germ-Line Development in the Nematode Caenorhabditis Elegans

To identify genes that encode maternal components required for development of the germ line in the nematode Caenorhabditis elegans, we have screened for mutations that confer a maternal-effect sterile or "grandchildless" phenotype: homozygous mutant hermaphrodites produced by heterozygous mothers are themselves fertile, but produce sterile progeny. Our screens have identified six loci, defined by 21 mutations. This paper presents genetic and phenotypic characterization of four of the loci. The majority of mutations, those in mes-2, mes-3 and mes-4, affect postembryonic germ-line development; the progeny of mutant mothers undergo apparently normal embryogenesis but develop into agametic adults with 10-1000-fold reductions in number of germ cells. In contrast, mutations in mes-1 cause defects in cytoplasmic partitioning during embryogenesis, and the resulting larvae lack germ-line progenitor cells. Mutations in all of the mes loci primarily affect the germ line, and none disrupt the structural integrity of germ granules. This is in contrast to grandchildless mutations in Drosophila melanogaster, all of which disrupt germ granules and affect abdominal as well as germ-line development.     

Highly accurate analysis of heterozygous loci bysingle cell PCR.

Single cell PCR is a powerful method for determining the genetic properties of individual cells. In the instance of heterozygous loci, however, preferential amplification of one allele can lead to allele drop out (ADO) of the other allele. Fortunately ADO does not occur in all amplifications, and is usually random when it does occur, with both alleles being equally susceptible to drop out. Therefore pooling of results from multiple independently amplified cells should greatly improve the analysis of diallelic loci, and the misdiagnosis rate of diallelic loci should decrease exponentially with the number of cells analysed. We have shown that this is true and that multiplex PCR allows for the simultaneous identification of a cell in a mixture of cells using microsatellite loci known to be informative, and accurate genotyping at other loci. This approach has practical applications to non-invasive prenatal diagnosis where small numbers of fetal cells in the presence of maternal cells must be both identified and genotyped at loci involved in genetic disease.     

Serum-free culture and characterization of renal epithelial cells isolated from human fetal kidneys of varying gestational age

Summary Cell cultures were initiated from seven human fetal kidneys that varied in gestational age from 90 days to newborn. The growth medium utilized was a 1:1 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 supplemented with selenium (5 ng/ml), insulin (5μg/ml), transferrin (5μg/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). For all the kidney isolates, initial cell attachment occurred within 12 h through multicell spheroids, and by 24 h a rapidly growing population of cells was obtained. Confluency was reached within 3 to 6 days. A combination of light microscopy, immunohistochemistry, and ultrastructural evaluation was utilized to characterize the resulting cultures as epithelial and homogeneous within each isolate and among the isolates. That is, regardless of gestational age of the fetal kidney used as starting material, an identical or highly similar population of cells was obtained. By light microscopy, the cultures were noted to form very few domes, the number being an indication of transport activity. However, ultrastructural examination revealed that the cells were noted to form domes composed of only a few cells or “micro-domes” that would not be visible by light microscopy. Within the micro-domes as well as other areas of the monolayer an apparent absence of tight junctions was noted by routine transmission electron microscopy. However, by freeze fracture analysis cells were shown to possess sealing strands, the structural component of tight junctions. It is postulated that the tight junctions of fetal epithelial cells are structurally altered as compared to tight junctions in adult renal epithelial cell cultures.     

Immune response of nestling warblers varies with extra-pair paternity and temperature

Extra-pair mating is widespread in birds, but its adaptive function remains unclear. It is often suggested that females obtain superior genes for their offspring as a consequence of extra-pair mating, but the evidence is limited. In this study, we examined the hypothesis that extra-pair mating provides females with offspring that have superior immune responses. We found that the T-cell-mediated immune response of extra-pair young was stronger than that of within-pair young in common yellowthroats (Geothlypis trichas). This paternity effect occurred when we compared all nestlings in the population, as well as in comparisons of both paternal and maternal half-siblings. Paternal half-siblings had a stronger immune response when they were produced with extra-pair females than with the male's social mate, which suggests that the greater immune response of extra-pair young was caused by nonadditive (compatible) genetic effects. However, these patterns were only significant in the colder of 2 years. Immune response was related positively to air temperature and nestlings had a stronger immune response in the warmer year. We suggest that such environmental variation could obscure the genetic benefits of extra-pair mating.     

Geographic variation in malarial parasite lineages in the common yellowthroat (<Emphasis Type="Italic">Geothlypis trichas</Emphasis>)

Our current understanding of migration routes of many birds is limited and researchers have employed various methods to determine migratory patterns. Recently, parasites have been used to track migratory birds. The objective of this study was to determine whether haemosporidian parasite lineages detect significant geographic structure in common yellowthroats (Geothlypis trichas). We examined liver tissue or blood from 552 birds sampled from multiple locations throughout the continental United States, southern Canada, and the Bahamas. We found a 52.7% overall prevalence of haematozoan infection. We identified 86.1% of these infections to genus: 81% were Plasmodium; 5% were Haemoproteus; and 0.1% were Leucocytozoon. There were significant differences in the prevalence of different parasite genera among regions (χ² = 36.82, P < 0.0001) and in the proportion of Plasmodium infections versus other parasites among regions (χ² = 35.52, P < 0.0001). Sequence information identified three Haemoproteus lineages, two Leucocytozoon lineages, and thirteen Plasmodium lineages. Due to the low number of Haemoproteus and Leucocytozoon, only Plasmodium lineages were used in the geographic comparison of lineages. Six Plasmodium lineages were found in eight or more birds and the prevalence of these varied significantly among regions (χ² = 172.33, P < 0.0001). Additionally, 45 juvenile birds were sampled to determine what parasites could be obtained in the breeding grounds and we found only one lineage. In conclusion, parasite lineages show some geographic structure, with some lineages being more geographically specific than others, but are not useful for determining migratory connectivity in this species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Epizootiology of Haemoproteus danilewskyi (Haemosporina: Haemoproteidae) in blue jays (Cyanocitta cristata) in southcentral Florida

Prevalence and density of Haemoproteus danilewskyi was studied in a population of free-ranging blue jays (Cyanocitta cristata) in southcentral Florida (USA) from May 1992 to December 1995. Prevalence of infection was 27% for data combined over years, seasons, ages, and sexes. Prevalence did not vary between sexes or among years, but increased with age and varied with season, being highest in June-July and lowest in November-January. Parasite density did not vary between sexes or among seasons, but was higher in younger birds when controlling for season. To determine periods of natural transmission, seasonal patterns of infection were compared with previous month abundance of the biting fly vectors. Mean monthly prevalence of H. danilewskyi in older jays was positively correlated with previous month abundance of Culicoides edeni and C. arboricola, both capable of sporogonic development of H. danilewskyi.