Transformation and allelic replacement in Francisella spp.

We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.     

Production of Hexagenia limbata nymphs in contaminated sediments in the Upper Great Lakes Connecting Channels

In April through October 1986, we sampled sediments and populations of nymphs of the burrowing mayfly, Hexagenia limbata (Serville), at 11 locations throughout the connecting channels of the upper Great Lakes, to determine if sediment contaminants adversely affected nymph production. Production over this period was high (980 to 9231 mg dry wt m^-2) at the five locations where measured sediment levels of oil, cyanide, and six metals were below the threshold criteria of the U.S. Environmental Protection Agency and the Ontario Ministry of Environment for contaminated or polluted sediments, and also where the criterion for visible oil given in the Water Quality Agreement between the U.S.A. and Canada for connecting waters of the Great Lakes was not exceeded. At the other six locations where sediments were polluted, production was markedly lower (359 to 872 mg dry wt m^-2). This finding is significant because it indicates that existing sediment quality criteria can be applied to protect H. limbata from oil, cyanide, and metals in the Great Lakes and connecting channels where the species fulfills a major role in secondary production and trophic transfer of energy.Contribution 733, of the National Fisheries Research Center-Great Lakes, U.S. Fish and Wildlife Service, 1451 Green Road, Ann Arbor, MI 48105.     

Preservation of Tracheal Mucus by Nonaqueous Fixative

Two nonaqueous fixatives, composed of fluorocarbon solvents with dissolved osmium tetroxide, were used to determine the feasibility of preserving the mucous coat in bovine and rat trachea for light and electron microscopy. Aqueous fixatives, while providing excellent cytological preservation, wash away the mucous lining, precluding ultrastructural analysis. Inclusion of ruthenium red or alcian blue within aqueous fixative improved retention of mucus, but provided incomplete, patchy results. Fixation with nonaqueous fluorocarbon solvent and dissolved osmium tetroxide preserved a continuous mucous epiphase layer above a clear hypophase layer. Subcomponents of the mucus included an electron dense surface layer, interrupted patches of mucus above the surface layer and electron dense membrane-like material within the mucus. This method of fixation will preserve mucus for light, scanning and transmission electron microscopy, using either intratracheal or immersion methods of fixation. The latter would enable use of materials from large animal models, autopsy or an abattoir.     

Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction

Summary The Ca²⁺ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the Junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m) [¹²⁵I]iodophenyl diazirine and then isolated and proteolysed with calpain II, the [¹²⁵I] was traced from the 565 kDa parent to M _r, 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa frament was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin     

Computer modelling of neural tube defects

Neurulation, the curling of the neuroepithelium to form the neural tube, is an essential component of the development of animal embryos. Defects of neural tube formation, which occur with an overall frequency of one in 500 human births, are the cause of severe and distressing congenital abnormalities. However, despite the fact that there is increasing information from animal experiments about the mechanisms which effect neural tube formation, much less is known about the fundamental causes of neural tube defects (NTD). The use of computer models provides one way of gaining clues about the ways in which neurulation may be compromised. Here we employ one computer model to examine the robustness of different cellular mechanisms which are thought to contribute to neurulation. The model, modified from that of Odell et al (Odell, G.M., Oster, G., Alberch, P. and Burnside, B., (1981)) mimics neurulation by laterally propagating a wave of apical contraction along an active zone within a ring of cells. We link the results to experimental evidence gained from studies of embryos in which neurulation has been perturbed. The results indicate that alteration of one of the properties of non-neural tissue can delay or inhibit neurulation, supporting the idea, gained from observation of embryos bearing genes which predispose to NTD, that the tissue underlying the neuroepithelium may contribute to the elevation of the neural folds. The results also show that reduction of the contractile properties of a small proportion of the neuroepithelial cell population may have a profound effect on overall tissue profiling. The results suggest that the elevation of the neural folds, and hence successful neurulation, may be vulnerable to relatively minor deficiencies in cell properties.     

Erythropoietic progenitor cells from premature neonates studied using a validated serum-deprived medium

We have examined a serum-deprived culture system in order to verify that it is suitable for the study of burst forming unit erythroid (BFU-E) progenitor cells from premature neonates. Optimum growth of BFU-E from premature neonates was observed with each media constituent using the same concentration as that previously described for adult subjects. Growth of immature BFU-E from premature neonates were highly dependant upon a source of Burst Promoting Activity and mature BFU-E derived colonies emerged at day 12 compared to day 14 in adults. Our preliminary results with the validated medium suggest that premature infants have increased peripheral blood concentrations of BFU-E compared to healthy adult controls.Abbreviations Ad Adherent cells - BPA Burst promoting activity - BFU-E Burst forming unit erythroid - Epo Erythropoietin - IL3 Interleukin-3 - LDC Low density (<1.077 g ml¹) peripheral blood mononuclear cells     

Migration in Archeology: The Baby and the Bathwater

Migration has been largely ignored by archeologists for the last two decades. Yet prehistoric demography and population studies are accepted as central concerns, and neither of these can be studied profitably without an understanding of migration. Recent books by Rouse and Renfrew have resurrected migration as a subject of serious analysis. It is proposed here that systems-oriented archeologists, in rejecting migration, have thrown out the baby with the bathwater. Traditional archeological approaches to migration fall short because a methodology for examining prehistoric migration must be dependent upon an understanding of the general structure of migration as a patterned human behavior. Aspects of such a structure are suggested and an application to a particular case in Eastern Europe is described.     

Immunofluorescence of sulphate-reducing bacteria

An indirect fluorescent antibody technique was used as a method of rapidly assessing and identifying sulphate-reducing bacteria. Five specific antisera and one polyvalent serum were raised and tested against 44 strains of the genera Desulfovibrio and Desulfotomaculum along with 4 control organisms. Immunofluorescence was found to be mainly strain specific with the sulphate-reducing bacteria although weak fluorescence was seen both within and between recognised groups. A polyvalent antiserum was successfully used to detect sulphate-reducing bacteria. No interference from 4 control organisms was found.