An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

Summary The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral micro-vasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

Phylogenetic inference: how much evolutionary history is knowable?

In order to reconstruct phylogenetic trees from extremely dissimilar sequences it is necessary to estimate accurately the extent of sequence divergence. In this paper a new method of sequence analysis, Markov triple analysis, is developed for determining the relative frequencies of nucleotide substitutions within the three branches of a three-taxon dendrogram. Assuming that nucleotide sites are independently and identically distributed and assuming a Markov model for nucleotide (or protein) evolution, it is shown that the unique Markov matrices can be reconstructed given only the joint probability distribution relating three taxa. (In the much simpler case involving only two taxa and two character states, Markov matrices can also be reconstructed, provided symmetry assumptions are placed on the elements of the matrices.) The method is illustrated using sequence data from the combined first and second codon positions derived from complete human, mouse, and cow mitochondrial sequences.      

DNA hybridization evidence for the principal lineages of hummingbirds (Aves:Trochilidae)

The spectacular evolutionary radiation of hummingbirds (Trochilidae) has served as a model system for many biological studies. To begin to provide a historical context for these investigations, we generated a complete matrix of DNA hybridization distances among 26 hummingbirds and an outgroup swift (Chaetura pelagica) to determine the principal hummingbird lineages. FITCH topologies estimated from symmetrized delta TmH-C values and subjected to various validation methods (bootstrapping, weighted jackknifing, branch length significance) indicated a fundamental split between hermit (Eutoxeres aquila, Threnetes ruckeri; Phaethornithinae) and nonhermit (Trochilinae) hummingbirds, and provided strong support for six principal nonhermit clades with the following branching order: (1) a predominantly lowland group comprising caribs (Eulampis holosericeus) and relatives (Androdon aequatorialis and Heliothryx barroti) with violet-ears (Colibri coruscans) and relatives (Doryfera ludovicae); (2) an Andean-associated clade of highly polytypic taxa (Eriocnemis, Heliodoxa, and Coeligena); (3) a second endemic Andean clade (Oreotrochilus chimborazo, Aglaiocercus coelestis, and Lesbia victoriae) paired with thorntails (Popelairia conversii); (4) emeralds and relatives (Chlorostilbon mellisugus, Amazilia tzacatl, Thalurania colombica, Orthorhyncus cristatus and Campylopterus villaviscensio); (5) mountain-gems (Lampornis clemenciae and Eugenes fulgens); and (6) tiny bee-like forms (Archilochus colubris, Myrtis fanny, Acestrura mulsant, and Philodice mitchellii). Corresponding analyses on a matrix of unsymmetrized delta values gave similar support for these relationships except that the branching order of the two Andean clades (2, 3 above) was unresolved. In general, subsidiary relationships were consistent and well supported by both matrices, sometimes revealing surprising associations between forms that differ dramatically in plumage and bill morphology. Our results also reveal some basic aspects of hummingbird ecologic and morphologic evolution. For example, most of the diverse endemic Andean assemblage apparently comprises two genetically divergent clades, whereas the majority of North American hummingbirds belong a single third clade. Genetic distances separating some morphologically distinct genera (Oreotrochilus, Aglaiocercus, Lesbia; Myrtis, Acestrura, Philodice) were no greater than among congeneric (Coeligena) species, indicating that, in hummingbirds, morphological divergence does not necessarily reflect level of genetic divergence.      

Effect of tyrosine kinase inhibition on basal and epidermal growth factor-stimulated human Caco-2 enterocyte sheet migration and proliferation

Mucosal healing requires enterocyte migration (restitution) supplemented by proliferation. Proliferation and migration may be studied independently by thymidine uptake and proliferation-blocked cell migration using human Caco-2 enterocyte monolayers in culture. Since epidermal growth factor (EGF) promotes mucosal healing and the EGF receptor is a tyrosine kinase, we hypothesized that tyrosine kinases might therefore modulate enterocyte migration and proliferation. The tyrosine kinase inhibitors genistein and 2, 5-dihydroxymethylcinnamate, which block kinase ATP-binding and substrate-binding sites, respectively, were studied alone and with EGF. Proliferation was blocked with mitomycin. Although each inhibitor decreased basal and EGF-stimulated monolayer expansion when cell proliferation occurred, neither genistein nor 2, 5-dihydroxymethylcinnamate decreased migration when proliferation was blocked. However, each inhibitor prevented EGF stimulation of proliferation-blocked migration and thymidine uptake. More substantial inhibition of basal proliferation by genistein correlated with increased protein-linked DNA breaks, which may reflect nonspecific inhibition of DNA topoisomerase activity by genistein. The more specific 2,5-dihydroxymeth-ylcinnamate blocked changes in the α2 integrin subunit organization which may modulate EGF-stimulated migration. Antiproliferative effects of tyrosine kinase inhibitors decrease basal monolayer expansion but true basal enterocyte migration appears independent of tyrosine kinase regulation. However, a specific tyrosine kinase-dependent modulation of cell-matrix interaction inhibits EGF-stimulated migration. © 1994 Wiley-Liss, Inc.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Differences in the carbon tetrachloride-induced damage to components of the smooth and rough endoplasmic reticulum from rat liver

There is a higher activity of ethyl morphine N-demethylase (EM-ase) and cytochrome P-450 (P-450) reductase as well as higher P-450 content in the smooth endoplasmic reticulum (SER) than in the rough endoplasmic reticulum (RER). The extent of the irreversible binding of the¹⁴C from¹⁴CCl₄ to lipids and proteins, as well as the CCl₄-induced destruction of P-450 is more intense in SER than in RER while the opposite was found for glucose 6-phosphatase (G6P-ase) destruction. CCl₄-induced lipid peroxidation is as intense in SER as is in RER.¹⁴C from¹⁴CCl₄ gets irreversibly bound to ribosomal proteins.     

Cancer cell binding to E-selectin transfected human endothelia.

Human endothelial cells were transiently transfected with E-Selectin which enabled us to study tumor cell/endothelial interactions following engagement of E-Selectin without the added complications of metabolic stimulation, morphological changes, and/or up regulation of other adhesion molecules due to cytokine induction. Similar results were received from in vitro binding studies and FACS analyses on both Tumor Necrosis Factor-alpha activated and E-Selectin transfected endothelial cells. These data suggest that this methodology is appropriate for dissecting the individual activities of E-selectin while minimizing the participation of other adhesion molecules, thereby allowing us to develop a better understanding of the role of E-Selectin and endothelia in metastatic disease.     

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

Background

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.

Results

The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.

Conclusion

This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-923) contains supplementary material, which is available to authorized users.