Isolation and properties in culture of human adrenal capillary endothelial cells

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.     

A Bird’s Eye View of Discard Reforms: Bird-Borne Cameras Reveal Seabird/Fishery Interactions

Commercial capture fisheries produce huge quantities of offal, as well as undersized and unwanted catch in the form of discards. Declines in global catches and legislation to ban discarding will significantly reduce discards, but this subsidy supports a large scavenger community. Understanding the potential impact of declining discards for scavengers should feature in an eco-system based approach to fisheries management, but requires greater knowledge of scavenger/fishery interactions. Here we use bird-borne cameras, in tandem with GPS loggers, to provide a unique view of seabird/fishery interactions. 20,643 digital images (one min⁻¹) from ten bird-borne cameras deployed on central place northern gannets Morus bassanus revealed that all birds photographed fishing vessels. These were large (>15 m) boats, with no small-scale vessels. Virtually all vessels were trawlers, and gannets were almost always accompanied by other scavenging birds. All individuals exhibited an Area-Restricted Search (ARS) during foraging, but only 42% of ARS were associated with fishing vessels, indicating much ‘natural’ foraging. The proportion of ARS behaviours associated with fishing boats were higher for males (81%) than females (30%), although the reasons for this are currently unclear. Our study illustrates that fisheries form a very important component of the prey-landscape for foraging gannets and that a discard ban, such as that proposed under reforms of the EU Common Fisheries Policy, may have a significant impact on gannet behaviour, particularly males. However, a continued reliance on ‘natural’ foraging suggests the ability to switch away from scavenging, but only if there is sufficient food to meet their needs in the absence of a discard subsidy.     

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

The pH-dependence of class B and class C beta-lactamases.

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.     

Micropropagation of Hieracium aurantiacum

Hieracium aurantiacum L. is being investigated as a model system for studying the genetic basis of gametophytic apomixis. Micropropagation methods were developed for the multiplication and maintenance of unique genotypes, and to facilitate the development of an Agrobacterium-mediated transformation procedure. A basal medium containing Murashige & Skoog macro- and micro-salts, B5 vitamins and 3% sucrose was used throughout. Shoot regeneration from leaf disks was optimized on a medium supplemented with 2.5 M indole-3-butyric acid (IBA) and 2.2 M 6-benzyladenine (BA). Shoot proliferation was optimized using 2.2 M BA, without additional IBA, giving 5-fold shoot-tip multiplication rates every 4 weeks. Shoots readily formed roots on the basal medium and could be transplanted to potting medium 3 weeks later. Micropropagation was found to have no effect on the expression of apomixis in this species.Abbreviations Ba 6-benzyladenine - iba indole-3-butyric acid - lsd least significant difference     

Selection for β2-microglobulin mutation in mismatch repair-defective colorectal carcinomas

Novel peptide antigens complexed with human leukocyte antigen (HLA) and β₂-microglobulin (β2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumour cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and β2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3] and [4] and loss of β2M (or HLA) expression [5] and [6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of β2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating β2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring β2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.     

Ds transposition mediated by transient transposase expression in Heiracium aurantiacum

The feasibility of using transient transposase expression to mobilize Ds elements for gene tagging in Hieracium aurantiacum was evaluated. A T-DNA construct carrying the Ac transposase gene and either a visible marker gene (uidA) or the conditionally-lethal marker gene (codA) was transferred to H. aurantiacum leaf discs (previously transformed with a Ds element) by co-cultivation with Agrobacterium tumefaciens. Shoots were regenerated directly from the co-cultivated leaf discs under selection for antibiotic resistance resulting from Ds excision. Most regenerants carried unique transposition events. Of 84 regenerated plants, twenty one (25%) did not express the marker gene and the DNA coding sequence of the transposase could not be detected in seven (8.3%). Potential advantages of this method over conventional gene-tagging methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; and the transient nature of transposase expression should facilitate the stabilisation of the transposed element.     

Post-construction avian mortality monitoring at Project West Wind

Abstract

Post-construction avifauna investigations were undertaken at Project West Wind, Meridian Energy Limited's 62-turbine wind farm on the Wellington south coast. These investigations were required in accordance with the resource consent conditions to quantify the level of avian mortalities occurring at the wind farm, particularly in regard to New Zealand falcon (Falco novaeseelandiae), kākā (Nestor meridionalis) and kererū (Hemiphaga novaeseelandiae). This is the first comprehensive study at a New Zealand operating wind farm. The methods included three field components necessary to calculate annual estimates of mortalities across the wind farm site: routine turbine searches; carcass detection trials; and carcass removal trials. Results from years 1 and 2 of a three-year programme are presented. To date, mortalities have been recorded for 17 taxa at 18 of the 24 study turbines. There have been no recorded mortalities of falcon, kākā or kererū. Australasian harrier (Circus approximans) has been the species for which the most mortalities have been recorded. Overall estimated annual mortality rates for years 1 and 2 were calculated to be approximately six and five birds per turbine respectively.     

Probable predation of Leach’s Storm‐petrel Oceanodroma leucorhoa eggs by St Kilda Field Mice Apodemus sylvaticus hirtensis

Capsule Leach’s Storm‐petrels Oceanodroma leucorhoa may be depredated by endemic St Kilda Field Mice Apodemus sylvaticus hirtensis.