P‐11THE IMPACT OF INTRODUCING LIQUID BASED CYTOLOGY INTO A ROUTINE SCREENING LABORATORY

Introduction: A glacial acetic acid wash performed retrospectively or prospectively on visibly bloodstained cervical ThinPrep® specimens can reduce the unsatisfactory rate and increase the number of diagnostic epithelial cells. This study was undertaken to determine which specimens are most likely to benefit from a prospective glacial acetic acid wash. Methods: Bloodstained ThinPrep® specimens selected for routine lysing prior to processing were macroscopically assessed and scored based on the level of blood present (+ to +++). Both unlysed and lysed slides were prepared from each specimen and microscopically examined. Results: Fifty‐eight specimens (32 scored +, 12 ++ and 14+++) were assessed. Three unlysed slides prepared from the ++ specimens and 12 from +++ specimens were evaluated as unsatisfactory due to excessive blood and inadequate numbers of squamous cells. In contrast, only one of the unlysed slides from the 32 + specimens was considered to be unsatisfactory. Almost all the lysed slides were satisfactory and generally more cellular than the unlysed slides. Abnormal cells were found in four cases (both unlysed and lysed paired slides). Discussion: Although the acetic acid wash increases cellularity of bloodstained ThinPrep® specimens, prospectively lysing all bloodstained specimens is an unnecessary procedure. Lysing only very heavily bloodstained specimens prior to processing will reduce laboratory workload, costs and the possibility of specimen mix up. Occasionally a retrospective wash may be required but screening staff should be aware that although blood may be present on an unlysed ThinPrep® slide, a lysed slide should not be requested unless there are insufficient numbers of squamous cells present.     

The influence of changing temperature on the life history of Daphniopsis ephemeralis

We present evidence which suggests that for Daphniopsis ephemeralischanges in water temperature may directly cue changes in sexratio, ephippial and parthenogenetic egg production and indirectlydetermine female body size. Field data suggested that over anannual cycle, a population of D. ephemeralis comprised animalswith three distinct life history phases. The first was madeup of exephippial females which hatched in the fall. They produceda second group made up of large parthenogenetic females whichcould produce more than one overwintering generation. In thespring these parthenogenetic females gave rise to a third lifehistory phase which comprised males and small ephippial females.Field enclosure experiments suggested that the small size ofspring females was not related to predation. Laboratory experimentssuggested that ephippial egg production at small body sizeswas associated with ‘increasing’ temperature. Theseexperiments also suggested that the fall reproductive pattern(parthenogenetic eggs and large body sizes) was associated with‘decreasing’ temperatures. We conclude that directionalityof seasonal temperature change may be the prime factor responsiblefor D. ephemeralis life history changes observed in the field.     

Involvement of the mutated M protein in altered budding polarity of a pantropic mutant, F1-R, of Sendai virus.

Wild-type Sendai virus buds at the apical plasma membrane domain of polarized epithelial MDCK cells, whereas a pantropic mutant, F1-R, buds at both the apical and basolateral domains. In F1-R-infected cells, polarized protein transport and the microtubule network are impaired. It has been suggested that the mutated F and/or M proteins in F1-R are responsible for these changes (M. Tashiro, J. T. Seto, H.-D. Klenk, and R. Rott, J. Virol. 67:5902-5910, 1993). To clarify which gene or mutation(s) was responsible for the microtubule disruption which leads to altered budding of F1-R, MDCK cell lines containing the M gene of either the wild type or F1-R were established. When wild-type M protein was expressed at a level corresponding to that synthesized in virus-infected cells, cellular polarity and the integrity of the microtubules were affected to some extent. On the other hand, expression of the mutated F1-R M protein resulted in the formation of giant cells about 40 times larger than normal MDCK cells. Under these conditions, the effects on the microtubule network were enhanced. The microtubules were disrupted and polarized protein transport was impaired as indicated by the nonpolarized secretion of gp80, a host cell glycoprotein normally secreted from the apical domain, and bipolar budding of wild-type and F1-R Sendai viruses. The mutated F glycoprotein of F1-R was transported bipolarly in cells expressing the F1-R M protein, whereas it was transported predominantly to the apical domain when expressed alone or in cells coexpressing the wild-type M protein. These findings indicate that the M protein of F1-R is involved in the disruption of the microtubular network, leading to impairment of cellular polarity, bipolar transport of the F glycoprotein, and bipolar budding of the virus.     

Predator-induced bottom-up effects in oligotrophic systems

Five treatments (replication n=2) were applied to mesocosms in an oligotrophic lake (TP=6–10 µg 1^-1) to assess the effects of fish on planktonic communities. The treatments were: (1) high fish (30 kg ha^–1 Lepomis auritus, Linnaeus), (2) low fish (10 kg ha^–1), (3) high removal of zooplankton, (4) low removal of zooplankton and (5) control. Total phosphorus, chlorophyll a, zooplankton biomass, and species richness decreased from high fish > low fish > control > low removal > high removal treatments. The fish treatments were dominated by crustacean zooplankton, while rotifers outnumbered the other zooplankters in the removal treatments. Calculations of zooplankton grazing rates suggested that clearance rates seldom exceeded 2% of the enclosure volume d^–1 and were unlikely to have had much influence on phytoplankton biomass. Calculations from a phosphorus bioenergetics model revealed that when fish were present, their excretion rates were higher than the rates ascribed to zooplankton. Diet analysis showed that the fish derived most of their energy from the benthos and periphyton, and that fish excretion and egestion made significant contributions to the very oligotrophic pelagic phosphorus pool. In the absence of fish, zooplankton excretion was highest in the control treatments and lowest in the zooplankton removal treatments. Our results suggest that in oligotrophic systems, planktivorous fish can be significant sources of phosphorus and that fish and zooplankton induced nutrient cycling have significant impacts on planktonic community structure.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

The PiGMaP consortium linkage map of the pig (Sus scrofa)

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (16.5 Morgans) than the genetic map of the homogametic sex (female) (21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be 18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.     

Effects of planktivore abundance on chlorophyll-a and Secchi depth

We used two analyses to test the hypothesis that planktivore abundances contribute to the residual variations of Secchi depth or chlorophyll-a plotted with respect to mean summer epilimnetic total phosphorus. The first analysis involved 15 lake years of data from six lakes. The data set comprised mark-recapture assessments of piscivore and planktivore numbers and estimates of mean summer chlorophyll-a, total phosphorus and Secchi depth. We found that residual chlorophyll-a variation was not significantly (p>0.05) correlated with planktivore densities, but that planktivore densities did contribute (p<0.02) to the residual variation of Secchi depth on mean total phosphorus. The second analysis included all of the data used in the first plus an additional 13 lake years of data from the literature. These data showed that the percentage of the total fish community comprising planktivores did not significantly (p>0.05) contribute to the residual variation in chlorophyll-a with respect to mean summer total phosphorus. Together, our results suggest that planktivore abundance has a significant cascading impact on water clarity, but no long term statistically significant impact on mean summer chlorophyll-a concentration.