Observer variation in quantification of immunocytochemistry by image analysis.

This paper reports the findings of a study designed to examine observer variation as a source of inaccuracy inherent in the use of computer-assisted image analysis to measure areas of stained tissue. The rat pituitary immunostained for prolactin and galanin was used as an example to estimate patterns of immunoreactivity exhibited by different cell types. Six observers, with differing experience, selected grey level threshold values on 40 fields of images of stained tissue making three repeats of each field. The 40 fields consisted of 20 serial pairs of colocalized fields, one immunostained for prolactin, the other for galanin. The 20 pairs consisted of four pairs from each of five animals. Analysis of observer variation in the selection of threshold values showed large differences in the within- and between-observer variation. Analysis of the components of variance in the estimation of the ratios of stained tissues showed that the major source of variation was the within-observer component. An additional experiment using two observers, where half of the images were compared to the original microscope images before setting threshold levels, showed that the opportunity to make a comparison did not reduce observer variation. It is suggested that any study which uses semi-automatic methods to segment regions of a digital image can benefit from an analysis of this kind so that the sources of variation can be determined to enable maximum discriminating power in future studies.     

Streptomyces griseus streptomycin phosphotransferase: expression of its gene in Escherichia coli and sequence homology with other antibiotic phosphotransferases and with eukaryotic protein kinases

The aphD gene of Streptomyces griseus, encoding a streptomycin 6-phosphotransferase (SPH), was sub-cloned in the pBR322-based expression vector pRK9 (which contains the Serratia marcescens trp promoter) with selection for expression of streptomycin resistance in Escherichia coli. Two hybrid plasmids, pCKL631 and pCKL711, were isolated which conferred resistance. Both contained a approximately 2 kbp fragment already suspected to include aphD. The properties of in vitro deletion derivatives of these plasmids were consistent with the presumed location of aphD. In vitro deletion of a sequence including most of the trp promoter largely, but not quite completely, abolished the ability of the plasmid to confer streptomycin resistance, confirming that expression was indeed principally from the trp promoter. A polypeptide of approximately 34.5 kDa was present in minicells containing plasmids that conferred streptomycin resistance, but was absent when the plasmids contained in vitro deletions removing streptomycin resistance. Part of the fragment was sequenced and an open reading frame corresponding to aphD identified. A computer-assisted comparison of the deduced SPH sequence with those of other antibiotic phosphotransferases suggested a common structure A-B-C-D-E, where B and D were conserved between all sequences compared while A, C and E divided between the streptomycin and hygromycin B phosphotransferases on one hand and kanamycin/neomycin ones on the other. A composite sequence data base was searched for homologues to consensus matrices constructed from five approximately 12-residue subsequences within blocks B and D. For one subsequence, corresponding to the N-terminal portion of block D, those sequences from the database that yielded the highest homology scores comprised almost entirely either antibiotic phosphotransferases or eukaryotic protein kinases. Possible evolutionary implications of this homology, previously described by other groups, are discussed.     

The Darwinian stickleback Gasterosteus aculeatus: a history of evolutionary studies*

The history of studies on the taxonomy and evolutionary biology of the three‐spined stickleback Gasterosteus aculeatus from the 18th century to the present is reviewed. After the publication of Darwin's Origin of the Species, four important dates, 1925, 1947, 1967 and 2001, are identified as marking major gains in the understanding of the evolution of the diversity in morphological, life‐history, physiological and behavioural traits that characterizes G. aculeatus. The period 1925–1970 led to the identification of the main themes of research: status and adaptive significance of lateral‐plate morphs; inter and intrapopulation trait variation in freshwater resident G. aculeatus and the adaptive significance of the variation. Between 1970 and 2001, these themes were investigated using variation observed particularly along the Pacific coast of the U.S.A. and Canada, notably in the Cook Inlet region of Alaska and the Haida Gwaii Archipelago. Studies on adaptive radiation and reproductive isolation in lacustrine, ecomorph pairs (limnetics and benthics) discovered in the Strait of Georgia region have been particularly productive. From 2001, the application of genomic studies to these problems began to open up the study of the relationships between genotype, phenotype and selective advantage to causal analysis.     

Glycyl-L-proline transport in rabbit enterocyte basolateral-membrane vesicles.

The properties of a peptide-transport system in rabbit enterocyte basolateral membrane were examined with glycyl-L-proline as the substrate. Basolateral-membrane vesicles prepared from rabbit proximal intestine were characterized in terms of both purity and orientation. Marker-enzyme assays show that the basolateral-membrane marker, ouabain-sensitive K(+)-activated phosphatase, is enriched 17-fold with respect to the initial homogenate. The activities of enzymes used as markers for other membranes and organelles are low, and contamination of the final membrane fraction with these is minimal. The use of immunoblotting techniques further confirms the absence of brush-border-membrane contamination. Proteins in the basolateral-membrane vesicle preparation gave no cross-reaction with antibodies against the 140 kDa antigen and the Na+/glucose-symport protein, markers specific to the brush-border membrane of the enterocyte. Conversely, antibodies raised against the classical basolateral-membrane marker, the RLA class I histocompatibility complex, reacted strongly with a 43 kDa basolateral-membrane protein. The orientation of the basolateral-membrane vesicles was shown to be predominantly inside-out on determination by two independent criteria. The uptake of [1-14C]glycyl-L-proline by these vesicles is stimulated by the presence of an inwardly directed pH gradient, and this stimulation can be abolished by the proton ionophores carbonyl cyanide p-trichloromethoxyphenylhydrazone (CCCP) and tetrachlorotrifluoromethylbenzimidazole (TTFB). Transport is also inhibited by HgCl2, thimerosal, Na+ and other glycyl dipeptides.     

Egg-release behaviour in Antarctic krill

The process of egg release is a complex and crucial step in the life cycle of euphausiids, especially with regards mortality and recruitment success. We examined this process in Antarctic krill (Euphausia superba) in terms of the functioning of the female genital apparatus and associated swimming behaviour. A tethering technique combined with video analysis was used to make observations of three females during the release process. We found eggs were steadily extruded over a period of up to 10 h, during which time the krill released between 1,600 and 4,000 eggs and the ovary reduced by a half in length and a third in height. Eggs were mainly released individually or, less commonly, in batches of between 2 and 4 eggs. Release rates were between 5 and 30 eggs min⁻¹. The steady release of eggs fits well with histological evidence that mature oocytes must pass individually through tight genital ducts with narrow apertures, before coming in to close contact with the sperm plug for fertilisation, and then expulsion from the thelycum as fertilised eggs. During spawning, the female alternated between slow and rapid rates of pleopod beating with egg release occurring at the moment of beat acceleration. At the point of release, the descent of the egg was accelerated through downward beats of the 7th thoracic leg. The cyclic pattern in pleopod beat-rate during spawning may alter swimming performance and contribute to the widely reported sex- and maturity-based biases within krill swarms.     

Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razor-shell (Ensis siliqua)

Much of the current knowledge concerning bivalve immunology and immunotoxicology has come from studies on the mussel genus, Mytilus, or from the American oyster, Crassostrea virginica. Following a major oil spill, it was observed that the marine mussel, Mytilus edulis, underwent significant immunosuppression but no oil-induced mortalities, while in contrast, mass mortalities were noted in the edible cockle, Cerastoderma edule, and the razor-shell, Ensis siliqua. A study comparing immune cells and functions in these three species was initiated (i) to assess whether M. edulis was a representative model species and (ii) to provide baseline data on immunity in two common species, which had previously received little or no attention in this respect. While all three species shared similar cell types, their lectin-binding and enzyme cytochemistry differed considerably. M. edulis had significantly different proportions of haemocytes binding with the lectins concanavalin A, wheatgerm agglutinin and Helix pomatia agglutinin and stained positive for eight enzymes, compared with only five in C. edule and three in E. siliqua. In terms of immune function, M. edulis haemocytes were much more active in phagocytosis and superoxide generation than haemocytes of the other two species. The results show that immune cells and functions differed extensively in these three closely related species, with M. edulis showing a much higher level of immunological vigour that may be linked to its considerable resilience to adverse environmental conditions. This suggests that M. edulis may not be particularly representative of the bivalves in terms of immune reactivity and that a wider range of species should be included in studies of molluscan immunotoxicology.